Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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DEVICE HAVING HORIZONTAL NANOCHANNEL FOR NANOPORE SEQUENCING
BACKGROUND
[0001] Some polynucleotide sequencing techniques involve
performing a large
number of controlled reactions on support surfaces or within predefined
reaction chambers.
The controlled reactions may then be observed or detected, and subsequent
analysis may help
identify properties of the polynucleotide involved in the reaction. Examples
of such
sequencing techniques include next-generation sequencing or massive parallel
sequencing
involving sequencing-by-ligation, sequencing-by-synthesis, reversible
terminator chemistry,
or pyrosequencing approaches.
[0002] Some polynucleotide sequencing techniques utilize a
nanopore, which can
provide a path for an ionic electrical current. For example, as the
polynucleotide traverses
through the nanopore, it influences the ionic current through the nanopore.
Each passing
nucleotide, or series of nucleotides, that passes through the nanopore yields
a characteristic
electrical current. These characteristic electrical currents as a result of
the traversing
polynucleotide can be recorded to determine the sequence of the
polynucleotide.
SUMMARY
100031 Provided in examples herein are devices for
sequencing biopolymers, e.g.,
polynucleotides, proteins, or peptides, methods of manufacturing the devices,
and methods of
using the devices.
[00041 In some embodiments, a nanopore sequencing device
is disclosed. In some
embodiments, the nanopore sequencing device comprises a substrate comprising a
dielectric
layer and at least one sensing electrode on a surface of the dielectric layer;
a cis well associated
with a cis electrode; a trans well associated with a trans electrode; a middle
well associated
with the sensing electrode and positioned on the substrate, wherein the middle
well is
positioned on the substrate and in fluid communication with the cis well and
the trans well; a
nanopore fluidically connecting the cis well and the middle well; and a
nanochannel fluidically
connecting the middle well and the trans well, wherein the nanochannel is
formed on the
surface of the substrate.
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[0005] The nanopore sequencing device of any of the
preceding embodiments,
wherein the nanochannel does not comprise a through-hole in the substrate.
[0006] The nanopore sequencing device of any of the
preceding embodiments,
wherein the nanopore is positioned in and through a membrane separating the
cis well and the
middle well.
[00071 The nanopore sequencing device of any of the
preceding embodiments,
wherein the membrane is formed of lipid, silicon, graphene, a solid-state
material, a synthetic
material, a biomimetic equivalent of lipid, or any combination thereof.
[0008] The nanopore sequencing device of any of the
preceding embodiments,
wherein the nanopore is a hollow in a structure formed of one or more
polynueleotides, one or
more polypeptides, one or more types of biopolymers, one or more carbon
nanotubes, one or
more types of solid-state materials, or any combination thereof disposed in
the membrane.
[0009] The nanopore sequencing device of any of the
preceding embodiments,
wherein the nanopore comprises biologically derived material.
MOM The nanopore sequencing device of any of the
preceding embodiments,
wherein the nanopore comprises a porin.
[00111 The nanopore sequencing device of any of the
preceding embodiments,
wherein the nanopore comprises non-biologically derived material.
[0012] The nanopore sequencing device of any of the
preceding embodiments,
wherein at least the cis well or the trans well is positioned horizontally
side-by-side with the
middle well.
[0013] The nanopore sequencing device of any of the
preceding embodiments,
wherein both the cis well and the trans well are positioned horizontally side-
by-side with the
middle well.
[0014] The nanopore sequencing device of any of the
preceding embodiments,
wherein the cis well is positioned horizontally side-by-side with the middle
well, and the trans
well is positioned vertically adjacent to the middle well.
[0015] The nanopore sequencing device of any of the
preceding embodiments,
wherein the trans well is positioned horizontally side-by-side with the middle
well, and the cis
well is positioned vertically adjacent to the middle well.
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[0016] The nanopore sequencing device of any of the
preceding embodiments,
wherein the middle well has a characteristic width of about 5 gm to about 200
1.1111.
[0017] The nanopore sequencing device of any of the
preceding embodiments,
wherein the middle well has a characteristic depth of about 5 gm to about 200
Rin.
[0018] The nanopore sequencing device of any of the
preceding embodiments,
wherein the cis well has a characteristic width of about 10 gm to about 10 mm.
[0019] The nanopore sequencing device of any of the
preceding embodiments,
wherein the trans well has a characteristic size of about 10 1.1111 to about
10 mm.
[00201 The nanopore sequencing device of any of the
preceding embodiments,
wherein the nanochannel has a tortuous path.
[0021] The nanopore sequencing device of any of the
preceding embodiments,
wherein the tortuous path comprises a rectangular wave shape, a sine wave
shape, a sawtooth
shape, a zipag shape, a spiral shape, or any combination thereof.
[0022] The nanopore sequencing device of any of the
preceding embodiments,
wherein th.e nanochannel has a path length that is chosen to achieve a desired
fluidic, ionic,
and/or electrical resistance.
[0023] The nanopore sequencing device of any of the
preceding embodiments,
wherein the nanochannel is about 5 nm to about 200 IIITI wide.
[0024] The nanopore sequencing device of any of the
preceding embodiments,
wherein the nanochannel has a footprint with a length of between about 5 p.m
and about 500
p.m.
[0025] The nanopore sequencing device of any of the
preceding embodiments,
wherein the path length of the nanochannel is about 1.5 to about 50 times the
length of the
nanochannel footprint
[0026] The nanopore sequencing device of any of the
preceding embodiments,
further comprising at least one bubble generator, at least one pressure pulse
generator, or any
combination thereof to control a liquid flow in at least one of the second
nanoscale openings.
[0027] The nanopore sequencing device of any of the
preceding embodiments,
further comprising: a plurality of middle wells, wherein each middle well is
associated with a
respective sensing electrode; each middle well is in fluid communication with
the cis well
through a respective nanopore; and each middle well is in fluid communication
with the trans
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well through a respective nanochannel, wherein the respective nanochannel is
oriented parallel
to the substrate surface.
[0028] The nanopore sequencing device of any of the
preceding embodiments,
wherein the respective nanopore is positioned in and through a respective
membrane separating
each of the middle wells and the cis well.
[0029] The nanopore sequencing device of any of the
preceding embodiments,
wherein the trans well is a common trans channel in fluid communication with
the plurality of
middle wells through respective nanochannels.
[0030] The nanopore sequencing device of any of the
preceding embodiments,
wherein the cis well is a common cis channel in fluid communication with the
plurality of
middle wells through respective nanopores.
[0031] The nanopore sequencing device of any of the
preceding embodiments,
wherein the middle wells are arranged in an ordered array.
[0032] The nanopore sequencing device of any of the
preceding embodiments,
wherein the device comprises at least 1,000,000 middle wells.
[0033] The nanopore sequencing device of any of the
preceding embodiments,
wherein the device further comprises a gas bubble generator configured to
generate a gas
bubble to modulate or block a flow of current, ions, and /or fluid in the
respective nanochannel.
[0034] The nanopore sequencing device of claim 29, wherein
the gas bubble
generator comprises the respective sensing electrode configured to generate
the gas bubble vis
electrolysis.
[0035] The nanopore sequencing device of any of the
preceding embodiments,
wherein the gas bubble generator comprises an electrode on the bottom of the
nanochannel
configured to generate the gas bubble via electrolysis or electrode wetting.
[0036] The nanopore sequencing device of any of the
preceding embodiments,
wherein the gas bubble generator comprises a resistive heater underneath the
nanochannel
configured to generate the gas bubble.
[0037] The nanopore sequencing device of any of the
preceding embodiments,
further comprises a gas bubble annihilator.
[0038] The nanopore sequencing device of any of the
preceding embodiments,
wherein the gas bubble annihilator comprises an actuator or a piezoelectric
element.
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[0039] In some embodiments, a method of manufacturing the
nanopore sequencing
device is disclosed. In some embodiments, the method comprises: providing a
first substrate
comprising a dielectric layer and at least one sensing electrode on a surface
of the first
substrate; forming at least one nanochannel on the surface of the first
substrate; and forming a
first patterned layer over the substrate, wherein the first patterned layer
comprises a trans well
adjacent to the at least one nanochannel and at least one middle well above
the sensing
electrode.
[0040] The method of any of the previous embodiments,
wherein the at least one
nanochannel is formed along the surface of the first substrate without forming
a through-hole
in the substrate.
[0041] The method of any of the previous embodiments,
wherein forming at least
one nanochannel comprises etching the nanochannel into the surface of the
first substrate.
[0042] The method of any of the previous embodiments,
wherein forming at least
one nanochannel comprises forming a patterned nanochannel structure on the
surface of the
first substrate.
[0043] The method of any of the previous embodiments,
wherein forming a first
patterned layer comprises depositing a patterning material layer over the
substrate, and
patterning the patterning material layer to expose the at least one sensing
electrode and
openings to the at least one nanochannel.
[0044] The method of any of the previous embodiments,
further comprising
forming an oxide or nitride layer in the at least one nanochannel, thereby
reducing the width
of the nanochannel.
[0045] The method of any of the previous embodiments,
further comprising:
depositing a capping layer over the first substrate prior to forming the first
patterned layer; and
patterning the capping layer to expose the at least one sensing electrode and
openings to the at
least one nanochannel.
[0046] The method of any of the previous embodiments,
wherein the trans well and
the middle well are positioned side-by-side on the first substrate.
[00471 The method of any of the previous embodiments,
wherein the first patterned
layer further comprises a cis well next to and positioned side-by-side to the
at least one middle
well.
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[0048] The method of any of the previous embodiments,
further comprising:
providing a second substrate have a second patterned layer attached; and
bonding the second
patterned layer with the first patterned layer, thereby further define the cis
well, the middle
Nvell, and the trans well between the first substrate and the second
substrate.
[0049] The method of any of the previous embodiments,
wherein the second
substrate further comprises fluidic inlet and/or outlet holes.
[00501 The method of any of the previous embodiments,
further comprising
introducine a membrane between the cis well and middle well.
[0051] The method of any of the previous embodiments,
wherein the membrane
between the cis well and middle well is a lipid membrane.
[0052] The method of any of the previous embodiments,
further comprising
depositing a protein into the membrane between the cis well and middle well,
thereby forming
nanopore through the membrane.
[0053] In some embodiments, another method of
manufacturing the nanopore
sequencing device is disclosed. In some embodiments, the method comprises
providing a first
substrate comprising a dielectric layer and at least one sensing electrode on
a surface of the
first substrate; forming at least one nanochannel on the surface of the first
substrate; depositing
a sacrificial material into the at least one nanochannel; forming a first
patterned layer over the
substrate, wherein the first patterned layer comprises a trans well adjacent
to the at least one
nanochannel and at least one middle well above the sensing electrode; and
removing the
sacrificial material, thereby opening the at least one nanochannel.
[0054] The method of any of the previous embodiments,
wherein the at least one
nanochannel is formed along the surface of the first substrate without forming
a through-hole
in the substrate.
[0055] The method of any of the previous embodiments,
wherein forming at least
one nanochannel comprises etching the nanochannel into the surface of the
first substrate.
10056) The method of any of the previous embodiments,
wherein forming at least
one nanochannel comprises forming a patterned nanochannel structure on the
surface of the
first substrate.
[0057] The method of any of the previous embodiments,
wherein forming a first
patterned layer comprises depositing a patterning material layer over the
substrate, and
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patterning the patterning material layer to expose the at least one sensing
electrode and
openings to the at least one nanochannel.
[0058] The method of any of the previous embodiments,
further comprising
forming an oxide or nitride layer in the at least one nanochannel, thereby
reducing the width
of the nanochannel.
[00591 The method of any of the previous embodiments,
further comprising
depositing a capping layer over the first substrate prior to forming the first
patterned layer; and
patterning the capping layer to expose the at least one sensing electrode and
openings to the at
least one nanochannel.
[0060] The method of any of the previous embodiments,
wherein the trans well and
the middle well are positioned side-by-side on the first substrate.
1-00611 The method of any of the previous embodiments,
wherein the first patterned
layer further comprises a cis well next to and positioned side-by-side to the
at least one middle
well.
[0062] The method of any of the previous embodiments,
further comprising
providing a second substrate have a second patterned layer attached; and
bonding the second
patterned layer with the first patterned layer, thereby further define the cis
well, the middle
well, and the trans well between the first substrate and the second substrate.
[00631 The method of any of the previous embodiments,
wherein the second
substrate further comprises fluidic inlet and/or outlet holes.
[0064] The method of any of the previous embodiments,
further comprising
introducing a membrane between the cis well and middle well.
[0065] The method of any of the previous embodiments,
wherein the membrane
between the cis well and middle well is a lipid membrane.
[0066] The method of any of the previous embodiments,
further comprising
depositing a protein into the membrane between the cis well and middle well,
thereby forming
a nanopore through the membrane.
[0067] In some embodiments, a method of manufacturing the
nanopore sequencing
device is disclosed. In some embodiments, the method comprises providing a
first substrate
comprising a dielectric layer and at least one sensing electrode on a surface
of the first
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substrate; forming a trans well in the dielectric layer; and forming at least
one nanochannel on
the surface of the first substrate between the trans well and the at least one
sensing electrode.
[0068] The method of any of the previous embodiments,
further comprising
depositing a pattering material layer over the substrate; and patterning the
patterning material
layer to form a patterned layer comprising at least one middle well above the
at least one
sensing electrode, wherein the middle well is in fluid communication with the
trans well
through the at least one nanochannel.
[0069] The method of any of the previous embodiments,
wherein the trans well is
a common trans well that is in fluid communication with a plurality of middle
wells through a
plurality of the nanochannels.
[0070] The method of any of the previous embodiments,
wherein the pattering
material layer comprises a dry film photoresist.
[0071] The method of any of the previous embodiments,
wherein the first patterned
layer further comprises a cis well next to and positioned side-by-side to the
at least one middle
well.
[0072] The systems, devices, kits, and methods disclosed
herein each have several
aspects, no single one of which is solely responsible for their desirable
attributes. Without
limiting the scope of the claims, some prominent fr.:it-tires will now be
discussed briefly.
Numerous other examples are also contemplated, including examples that have
fewer,
additional, and/or different components, steps, features, objects, benefits,
and advantages. The
components, aspects, and steps may also be arranged and ordered differently.
After
considering this discussion, and particularly after reading the section
entitled "Detailed
Description," one will understand how the features of the devices and methods
disclosed herein
provide advantages over other known devices and methods.
[0073] It is to be understood that any features of the
device and/or of the array
disclosed herein may be combined together in any desirable manner and/or
configuration.
Further, it is to be understood that any features of the method of using the
device may be
combined together in any desirable manner. Moreover, it is to be understood
that any
combination of features of this method and/or of the device and/or of the
array may be used
together, and/or may be combined with any of the examples disclosed herein.
Still further, it
is to be understood that any feature or combination of features of any of the
devices and/or of
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the arrays and/or of any of the methods may be combined together in any
desirable manner,
and/or may be combined with any of the examples disclosed herein.
[0074] It should be appreciated that all combinations of
the foregoing concepts and
additional concepts discussed in greater detail below are contemplated as
being part of the
inventive subject matter disclosed herein and may be used to achieve the
benefits and
advantages described herein.
BRIEF DESCRIPTION OF THE DRAWINGS
100751 Features of examples of the present disclosure will
become apparent by
reference to the following detailed description and drawings, in which like
reference numerals
correspond to similar, though perhaps not identical, components. For the sake
of brevity,
reference numerals or features having a previously described function may or
may not be
described in connection with other drawings in which they appear.
[00761 FIG. 1A is a cross-sectional side view of an
example nanopore sequencing
device.
[00771 FIG. 1B illustrates another example for generating
water electrolysis.
[00781 FIG. 2 shows a schematic circuit diagram of the
electrical resistance
provided by the nanopore sequencing device of FIG. 1A.
[0079] FIG. 3A is a cross-sectional top view of the
nanopore sequencing device of
FIG. 1A.
[0080] FIG. 3B is a cross-sectional top view of the
nanopore sequencing device of
FIG. 1A. having an alternative nanochannel structure.
[0081] FIG. 4 is a cross-sectional top view of an example
sequencing system
utilizing the nanopore sequencing device of FIG. IA.
[0082] FIG. 5A to FIG. 5M illustrate an example process
flow of manufacturing a
nanopore sequencing device.
[0083] FIG. 6 illustrates yet another example nanopore
sequencing device which
can generate water electrolysis.
[0084] FIG. 7A illustrates an example nanopore sequencing
device with a resistive
heater.
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[0085] FIG. 7B is a cross-sectional top view of another
example sequencing system
having a pair of electrodes for generating water electrolysis.
[0086] FIG. 8 is a cross-sectional top view of another
example sequencing system
utilizing the nanopore sequencing device of FIG. IA.
[0087] FIG. 9A illustrates a cross-sectional view of a
portion of an alternative
embodiment of a nanopore sequencing device where membranes are formed
horizontally.
[0088] FIG. 9B illustrates a top view of an alternative
embodiment of a nanopore
sequencing device where membranes are formed horizontally.
[0089] FIG. 9C illustrates another top view of an
alternative embodiment of a
nanopore sequencing device where membranes are formed horizontally.
[0090] FIG. 10 is a cross-sectional view of a portion of
another embodiment of a
nanopore sequencing device showing the relative positions of the trans well
and the middle
wel I
DETAILED DESCRIPTION
[0091] All patents, applications, published applications
and other publications
referred to herein are incorporated herein by reference to the referenced
material and in their
entireties. If a term or phrase is used herein in a way that is contrary to or
otherwise
inconsistent with a definition set forth in the patents, applications,
published applications and
other publications that are herein incorporated by reference, the use herein
prevails over the
definition that is incorporated herein by reference.
Definitions
(00921 All technical and scientific terms used herein have
the same meaning as
commonly understood to one of ordinary skill in the art to which this
disclosure belongs unless
clearly indicated otherwise.
[0093] As used herein, the singular forms "a", "and", and
"the" include plural
referents unless the context clearly dictates otherwise. Thus, for example,
reference to "a
sequence" may include a plurality of such sequences, and so forth.
[0094] The terms comprising, including, containing and
various forms of these
terms are synonymous with each other and are meant to be equally broad.
Moreover, unless
explicitly stated to the contrary, examples comprising, including, or having
an element or a
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plurality of elements having a particular property may include additional
elements, whether or
not the additional elements have that property.
[0095] As used herein, the terms "fluidically connecting,"
"fluid communication,"
"fluidically coupled," and the like refer to two spatial regions being
connected together such
that a fluid (e.g., liquid or gas) may flow between the two spatial regions.
For example, a cis
well/wells may be fluidically connected to a trans well/wells by way of a
middle well and/or a
nanochannel, such that a fluid, e.g., at least a portion of an electrolyte,
may flow between the
connected wells.
[0096] As used herein, the term "ionic connection" and the
like refer to two spatial
regions being connected together such that certain species of ions may flow
between the two
spatial regions.
[0097] As used herein, the term "electric connection" and
the like refer to two
spatial regions being connected together such that electrons, holes, ions or
other charge carriers
may flow between the two spatial regions.
[0098] If an electrolyte flows between two connected
wells, ions and electric
currents may also flow between the connected wells. In some examples, two
spatial regions
may be in fluid/ionic/electric communication through first and second
nanoscale openings, or
through one or more valves, restrictors, or other fluidic components that are
to control or
regulate a flow of fluid, ions or electric current through a system.
[0099] As used herein, the term "operably connected"
refers to a configuration of
elements, wherein an action or reaction of one element affects another
element, but in a manner
that preserves each element's functionality.
[0100] As used herein, the term "membrane" refers to a non-
permeable or semi-
permeable barrier or other sheet that separates two liquid/gel chambers (e.g.,
a cis well and a
fluidic cavity or reservoir) which can contain the same compositions or
different compositions
therein. The permeability of the membrane to any given species depends upon
the nature of
the membrane. In some examples, the membrane may be non-permeable to ions, to
electric
current, and/or to fluids. For example, a lipid membrane may be impermeable to
ions (i.e.,
does not allow any ion transport therethrough), but may be at least partially
permeable to water
(e.g., water diffusivity ranges from about 40 !mils to about 100 m/s). For
another example,
a synthetic/solid-state membrane, one example of which is silicon nitride, may
be impermeable
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to ions, electric charge, and fluids (i.e., the diffusion of all of these
species is zero). Any
membrane may be used in accordance with the present disclosure, as long as the
membrane
can include a transmembrane nanoscale opening and can maintain a potential
difference across
the membrane. The membrane may be a monolayer or a multilayer membrane. A
multilayer
membrane includes two or more layers, each of which is a. non-permeable or
semi-permeable
material.
[0101] The membrane may be formed of materials of
biological or non-biological
origin. A material that is of biological origin refers to material derived
from or isolated from
a biological environment such as an organism or cell, or a synthetically
manufactured version
of a biologically available structure (e.g., a biomimetic material).
[0102] An example membrane that is made from the material
of biological origin
includes a monolayer formed by a bolalipid. Another example membrane that is
made from
the material of biological origin includes a lipid bilayer Suitable lipid
bilayers include, for
example, a membrane of a cell, a membrane of an organelle, a liposome, a
planar lipid bilayer,
and a supported lipid bilayer. A lipid bilayer can be formed, for example,
from two opposing
layers of phospholipids, which are arranged such that their hydrophobic tail
groups face
towards each other to form a hydrophobic interior, whereas the hydrophilic
head groups of the
lipids face outwards towards the aqueous environment on each side of the
bilayer. Lipid
bilayers also can be formed, for example, by a method in which a lipid
monolayer is carried
on an aqueous solution/air interface past either side of an aperture that is
substantially
perpendicular to that interface. The lipid is normally added to the surface of
an aqueous
electrolyte solution by first dissolving it in an organic solvent and then
allowing a drop of the
solvent to evaporate on the surface of the aqueous solution on either side of
the aperture. Once
the organic solvent has at least partially evaporated, the solution/air
interfaces on either side of
the aperture are physically moved up and down past the aperture until a
bilayer is formed.
Other suitable methods of bilayer formation include tip-dipping, painting
bilayers, and patch-
clamping of liposome bilayers. Any other methods for obtaining or generating
lipid bilayers
may also be used.
[01031 A material that is not of biological origin may
also be used as the membrane.
Some of these materials are solid-state materials and can form a solid-state
membrane, and
others of these materials can form a thin liquid film or membrane. The solid-
state membrane
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can be a monolayer, such as a coating or film on a supporting substrate (i.e.,
a solid support),
or a freestanding element. The solid-state membrane can also be a composite of
multilayered
materials in a sandwich configuration. Any material not of biological origin
may be used, as
long as the resulting membrane can include a transmembrane nanoscale opening
and can
maintain a potential difference across the membrane. The membranes may include
organic
materials, inorganic materials, or both. Examples of suitable solid-state
materials include, for
example, microelectronic materials, insulating materials (e.g., silicon
nitride (Si3N4),
aluminum oxide (A1203), hafnium oxide (Hf02), tantalum pentoxide (Ta2O5),
silicon oxide
(SiO2), etc.), some organic and inorganic polymers (e.g., polyamide, plastics,
such as
polytetrafluoroethylene (PTFE), or elastomers, such as two-component addition-
cure silicone
rubber), and glasses. In addition, the solid-state membrane can be made from a
monolayer of
graphene, which is an atomically thin sheet of carbon atoms densely packed
into a two-
dimensional honeycomb lattice, a multilayer of graphene, or one or more layers
of graphene
mixed with one or more layers of other solid-state materials. A graphene-
containing solid-
state membrane can include at least one graphene layer that is a graphene
nanoribbon or
graphene nanogap, which can be used as an electrical sensor to characterize
the target
polynucleotide. It is to be understood that the solid-state membrane can be
made by any
suitable method, for example, chemical vapor deposition (CVD). In an example,
a graphene
membrane can be prepared through either CVD or exfoliation from graphite.
Examples of
suitable thin liquid film materials that may be used include diblock
copolymers or triblock
copolymers, such as amphiphilic PIVIOXA-PDMS-PMOXA ABA triblock copolymers.
[0104] As used herein, the term "nanopore" is intended to
mean a hollow structure
discrete from, or defined in, and extending across the membrane. The nanopore
permits ions,
electric current, and/or fluids to cross from one side of the membrane to the
other side of the
membrane. For example, a membrane that inhibits the passage of ions or water-
soluble
molecules can include a nanopore structure that extends across the membrane to
permit the
passage (through a nanoscale opening extending through the nanopore structure)
of the ions or
water-soluble molecules from one side of the membrane to the other side of the
membrane.
The diameter of the nanoscale opening extending through the nanopore structure
can vary
along its length (i.e., from one side of the membrane to the other side of the
membrane), but at
any point is on the nanoscale (i.e., from about 1 nm to about 100 nm, or to
less than 1000 nm).
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Examples of the nanopore include, for example, biological nanopores, solid-
state nanopores,
and biological and solid-state hybrid nanopores.
[0105] As used herein, the term "diameter" is intended to
mean a longest straight
line inscribable in a cross-section of a nanoscale opening through a centroid
of the cross-
section of the nanoscale opening. It is to be understood that the nanoscale
opening may or may
not have a circular or substantially circular cross-section. Further, the
cross-section may be
regularly or irregularly shaped.
[0106] As used herein, the term "biological nanopore" is
intended to mean a
nanopore whose structure portion is made from materials of biological origin.
Biological
origin refers to a material derived from or isolated from a biological
environment such as an
organism or cell, or a synthetically manufactured version of a biologically
available structure.
Biological nanopores include, for example, polypeptide nanopores and poly-
nucleotide
nanopores.
[0107] As used herein, the term "polypeptide nanopore" is
intended to mean a
protein/polypeptide that extends across the membrane, and permits ions,
electric current,
biopolymers such as DNA. or peptides, or other molecules of appropriate
dimension and
charge, and/or fluids to flow therethrough from one side of the membrane to
the other side of
the membrane. A polypeptide nanopore can be a monomer, a homopolymer, or a
heteropolymer. Structures of polypeptide nanopores include, for example, an a-
helix bundle
nanopore and a 0-barrel nanopore. Example polypeptide nanopores include a-
hemolysin,
Mycobacterium smegmatis porin A (MspA), gramicidin A, maltoporin, OmpF, OmpC,
PhoE,
Tsx, F-pilus, aerolysin, etc. The protein a-hemolysin is found naturally in
cell membranes,
where ii. acts as a pore for ions or molecules to be transported in and out of
cells.
Mycobactertutn smegmatis porin A (MspA) is a membrane porin produced by
Mycobacteria,
which allows hydrophilic molecules to enter the bacterium. MspA forms a
tightly
interconnected octamer and tratismernbrane beta-barrel that resembles a goblet
and contains a
central pore.
[0108] A polypeptide nanopore can be synthetic. A
synthetic polypeptide
nanopore includes a protein-like amino acid sequence that does not occur in
nature. The
protein-like amino acid sequence may include some of the amino acids that are
known to exist
but do not form the basis of proteins (i.e., non-proteinogenic amino acids).
The protein-like
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amino acid sequence may be artificially synthesized rather than expressed in
an organism and
then purified/isolated.
[0109] As used herein, the term "polynucleotide nanopore"
is intended to include
a polynucleotide that extends across the membrane, and permits ions, electric
current, and/or
fluids to flow from one side of the membrane to the other side of the
membrane. A
polynucleotide pore can include, for example, a polynucleotide origami (e.g.,
nanoscale
folding of DNA to create the nanopore).
[0110] Also as used herein, the term "solid-state
nanopore" is intended to mean a
nanopore whose structure portion is defined by a solid-state membrane and
includes materials
of non-biological origin (i.e., not of biological origin). A solid-state
nanopore can be formed
of an inorganic or organic material. Solid-state nanopores include, for
example, silicon nitride
nanopores, silicon dioxide nanopores, and graphene nanopores.
[0111] The nanopores disclosed herein may be hybrid
nanopores. A "hybrid
nanopore" refers to a nanopore including materials of both biological and non-
biological
origins. An example of a hybrid nanopore includes a polypeptide-solid-state
hybrid nanopore
and a polynucleotide-solid-state nanopore.
[0112] In some embodiments, the nanopore may comprise a
solid-state material,
such as silicon nitride, modified silicon nitride, silicon, silicon oxide, or
graphene, or a
combination thereof In some embodiments, the nanopore is a protein that forms
a tunnel upon
insertion into a bilayer, membrane, thin film, or solid-state aperture. In
some embodiments, the
nanopore is comprised in a lipid bilayer. In some embodiments, the nanopore is
comprised in
an artificial membrane comprising a mycolic acid. The nanopore may be a
Myeabacierium
smegmatis porin (Msp) having a vestibule and a constriction zone that define
the tunnel. The
Msp porin may be a mutant MspA porin. In some embodiments, amino acids at
positions 90,
91, and 93 of the mutant MspA porin are each substituted with asparagine. Some
embodiments
may comprise altering the translocation velocity or sequencing sensitivity by
removing,
adding, or replacing at least one amino acid of an Msp porin. A "mutant MspA
porin" is a
multimer complex that has at least or at most 70, 75, 80, 85, 90, 95, 98, or
99 percent or more
identity, or any range derivable therein, but less than 100%, to its
corresponding wild-type
MspA porin and retains tunnel-forming capability. A mutant MspA porin may be
recombinant
protein. Optionally, a mutant MspA porin is one having a mutation in the
constriction zone or
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the vestibule of a wild-type MspA porin. Optionally, a mutation may occur in
the rim or the
outside of the periplasmic loops of a wild-type N1spA porin. A mutant MspA
porin may be
employed in any embodiment described herein.
[01131 A "vestibule" refers to the cone-shaped portion of
the interior of an Msp
porin whose diameter generally decreases from one end to the other along a
central axis, where
the narrowest portion of the vestibule is connected to the constriction zone.
A vestibule may
also be referred to as a "goblet." The vestibule and the constriction zone
together define the
tunnel of an Msp porin. A "constriction zone" or the "readhead" refers to the
narrowest portion
of the tunnel of an Msp porin, in terms of diameter, that is connected to the
vestibule. The
length of the constriction zone may range from about 0.3 am to about 2 nrn.
Optionally, the
length is about, at most about, or at least about 0.3, 0.4, 0.5, 0.6, 0.7,
0.8, 0.9, 1.0, 1.1, 1.2, 1.3,
1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, or 3 nm, or any range derivable therein. The
diameter of the
constriction zone may range from about 0.3 nm to about 2 nrri. Optionally, the
diameter is
about, at most about, or at least about 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9,
1.0, 1.1, 1.2,1.3, 1.4,1.5,
1.6, 1.7, 1.8, 1.9, 2, or 3 nm, or any range derivable therein. A. "tunnel"
refers to the central,
empty portion of an. Msp porin that is defined by the vestibule and the
constriction zone,
through which a gas, liquid, ion, or analyte may pass. A tunnel is an example
of an opening of
a nanopore.
[0114] Various conditions such as light and the liquid
medium that contacts a
nanopore, including its pH, buffer composition, detergent composition, and
temperature, may
affect the behavior of the nanopore, particularly with respect to its
conductance through the
tunnel as well as the movement of an analyte with respect to the tunnel,
either temporarily or
permanently.
[0115] In some embodiments, the disclosed system for
nanopore sequencing
comprises an Msp porin having a vestibule and a constriction zone that define
a tunnel, wherein
the tunnel is positioned between a first liquid medium and a second liquid
medium, wherein at
least one liquid medium comprises an analyte polynucleotide, and wherein the
system is
operative to detect a property of the analyte. The system may be operative to
detect a property
of any analyte comprising subjecting an Msp porin to an electric field such
that the analyte
interacts with the Msp porin. The system may be operative to detect a property
of the analyte
comprising subjecting the Msp porin to an electric field such that the analyte
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electrophoretically translocates through the tunnel of the Msp porin. In some
embodiments,
the system comprises an Msp porin having a vestibule and a constriction zone
that define a
tunnel, wherein the tunnel is positioned in a lipid bilayer between a first
liquid medium and a
second liquid medium, and wherein the only point of liquid communication
between the first
and second liquid media occurs in the tunnel. Moreover, any Msp porin
described herein may
be comprised in any system described herein.
[0116] The system may further comprise one or more
temperature regulating
devices in communication with the fluid or electrolyte. The system described
herein may be
operative to translocate an analyte through an Msp porin tunnel either
electrophoretically or
otherwise.
[0117] As used herein, the term "nanopore sequencer"
refers to any of the devices
disclosed herein that can be used for nanopore sequencing. In the examples
disclosed herein,
during na.nopore sequencing, the nanopore is immersed in example(s) of the
electrolyte
disclosed herein and a potential difference is applied across the membrane. In
an example, the
potential difference is an electric potential difference or an electrochemical
potential
difference. An electrical potential difference can be imposed across the
membrane via a
voltage source that injects or administers current to at least one of the ions
of the electrolyte
contained in the cis well or one or more of the trans wells. An
electrochemical potential
difference can be established by a difference in ionic composition of the cis
and trans wells in
combination with an electrical potential. The different ionic composition can
be, for example,
different ions in each well or different concentrations of the same ions in
each well.
[0118] The application of the potential difference across
a nanopore may force the
franslocation of a nucleic acid through the nanopore. One or more signals are
generated that
correspond to the translocation of the nucleotide through the nanopore.
Accordingly, as a
target polynucleotide, or as a mononucleotide or a probe derived from the
target polynucleotide
or mononucleotide, transits through the nanopore, the current across the
membrane changes
due to base-dependent (or probe dependent) blockage of the constriction, for
example. The
signal from that change in current can be measured using any of a variety of
methods. Each
signal is unique to the species of nucleotide(s) (or probe) in the nanopore,
such that the resultant
signal can be used to determine a characteristic of the polynucleotide. For
example, the identity
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of one or more species of nucleotide(s) (or probe) that produces a
characteristic signal can be
determined.
[0119] As used herein, a "reporter" is composed of one or
more reporter elements.
Reporters include what are known as "tags" and "labels." Reporters serve to
parse the genetic
information of the target nucleic acid. "Encode" or "parse" are verbs
referring to transferring
from one format to another, and refers to transferring the genetic information
of target template
base sequence into an arrangement of reporters.
[0120] As used herein, a "peptide" refers to two or more
amino acids joined
together by an amide bond (that is, a "peptide bond"). Peptides comprise up to
or include 50
amino acids. Peptides may be linear or cyclic. Peptides may be a, 13, 7, 5, or
higher, or mixed.
Peptides may comprise any mixture of amino acids as defined herein, such as
comprising any
combination of D, L, a, 13, 7, 5, or higher amino acids.
[0121] As used herein, a "protein" refers to an amino acid
sequence having 51 or
more amino acids.
[0122] As used herein, a "poly merase" is an enzyme
generally used for joining 3'-
OH. N-triphosphate nucleotides, oligomers, and their analogs. Polymerases
include, but are not
limited to, DNA-dependent DNA polymerases, DNA-dependent RNA polymerases. RNA-
dependent DNA polymerases, RNA-dependent RNA polymerases, 17 DNA. polymerase,
T3
DNA polymerase, T4 DNA. polymerase, T7 RNA polymerase, T3 RNA polymerase, SP6
RNA
polymerase, DNA polymerase I, Klenow fragment, Thermophilus aquaticus DNA
polymerase,
Tth DNA polymerase, VentRe DNA polymerase (New England Biolabs), Deep VentRX
DNA
polymerase (New England Biolabs), Bst DNA Polymerase Large Fragment, Stoeffel
Fragment, 90N DNA Polymerase, 90N DNA polymerase, Pfu DNA Polymerase, Tfl DNA
Polymerase, Tth DNA Polymerase, RepliPHI Phi29 Polymerase, Tli DNA polymerase,
eukaryotic DNA polymerase beta, telomerase, TherminatorTm polymerase (New
England
Biolabs), KOD H1FiTM DNA polymerase (Novagen), KOD1 DNA polymerase, Q-beta
replicase, terminal transferase, AMV reverse transcriptase, M-MLV reverse
transcriptase, Phi6
reverse transcriptase, HIV- 1 reverse transcriptase, novel polymerases
discovered by
bioprospecting, and polymerases cited in US 2007/0048748, US 6,329,178, US
6,602,695, and
US 6,395,524 (incorporated by reference). These polymerases include wild-type,
mutant
isoforms, and genetically engineered variants.
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[0123]
As used herein, a "nucleotide" includes a nitrogen containing
heterocyclic
base, a sugar, and one or more phosphate groups. Nucleotides are monomeric
units of a nucleic
acid sequence.
Examples of nucleotides include, for example, ribonucleotides or
deoxyribonucleotides.
In ribonucleotides (RNA), the sugar is a ribose, and in
deoxyribonucleotides (DNA), the sugar is a deoxyribose, i.e., a sugar lacking
a hydroxyl group
that is present at the 2' position in ribose. The nitrogen containing
heterocyclic base can be a
purine base or a pyrimidine base. Purine bases include adenine (A) and guanine
(G), and
modified derivatives or analogs thereof. Pyrimidine bases include cytosine
(C), thymine (T),
and uracil (U), and modified derivatives or analogs thereof. The C-1 atom of
deoxyribose is
bonded to N-1 of a pyrimidine or N-9 of a purine. The phosphate groups may be
in the mono-
, di-, or tri-phosphate form. These nucleotides are natural nucleotides, but
it is to be further
understood that non-natural nucleotides, modified nucleotides or analogs of
the
aforementioned nucleotides can also be used.
[0124]
As used herein, "nucleobase" is a heterocyclic base such as adenine,
guanine, cytosine, thymine, uracil, inosine, xanthine, hypoxanthine, or a
heterocyclic
derivative, analog, or tautomer thereof. A nucleobase can be naturally
occurring or synthetic.
Non-limiting examples of nucleobases are adenine, guanine, thymine, cytosine,
uracil,
xanthine, hypoxanthine, 8-az.apurine, purines substituted at the 8 position
with methyl or
bromine, 9-oxo-N6-methyladenine, 2-aminoadenine, 7-dvaz.axanthine, 7-
dearaguanine, 7-
deaza-adenine, N4-ethanocytosine, 2,6- diaminopurine, N6-ethano-2,6-
diaminopurine, 5-
methylcytosine, 5-(C3-C6)- alkynylcytosine, 5-fluorouracil, 5-bromouracil,
thiouracil,
pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridine, isocytosine,
isoguanine, inosine,
7,8-dimethylalloxazine, 6-dihydrothymine, 5,6-dihydrouracil, 4-methyl-indole,
ethenoadenine
and the non-naturally occurring nucleobases described in U.S. Pat. Nos.
5,432,272 and
6,150,510 and PCT applications WO 92/002258, WO 93/10820, WO 94/22892, and WO
94/24144, and Fasman ("Practical Handbook of Biochemistry and Molecular
Biology", pp.
385-394, 1989, CRC Press, Boca Raton, LO), all herein incorporated by
reference in their
entireties.
[0125]
The term "nucleic acid" or "polynucleotide" refers to a
deoxyribonucleotide
or ribonucleotide polymer in either single- or double-stranded form, and
unless otherwise
limited, encompasses known analogs of natural nucleotides that hybridize to
nucleic acids in
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manner similar to naturally occurring nucleotides, such as peptide nucleic
acids (PNAs) and
phosphorothioate DNA. Unless otherwise indicated, a particular nucleic acid
sequence
includes the complementary sequence thereof. Nucleotides include, but are not
limited to,
ATP, dATP, CT!', dCTP, GTP, dGTP, UT!',
dUTP, 5-methyl-CTP, 5-methyl-dCTP, ITP,
dITP, 2-amino-adenosine-TI', 2-amino-deoxyadenosine-TP, 2-thiothymidine
triphosphate,
pyrrolo-pyrimidine triphosphate, and 2-thiocytidine, as well as the
alphathiotriphosphates for
all of the above, and 2'-0-methyl-ribonucleotide triphosphates for all the
above bases.
Modified bases include, but are not limited to, 5-Br-UT!', 5-Br-dUTP, 5-F-UTP,
5-F-dUTP,
5-propynyl dCTP, and 5-propynyl-dUTP.
[01261
For example, a template polynucleotide chain may be any sample that is
to
be sequenced, and may be composed of DNA, RNA, or analogs thereof (e.g.,
peptide nucleic
acids). The source of the template (or target) polynucleotide chain can be
genomic DNA,
messenger RNA, or other nucleic acids from native sources. In some cases, the
template
polynucleotide chain that is derived from such sources can be amplified prior
to use. Any of a
variety of known amplification techniques can be used including, but not
limited to,
polymerase chain reaction (PCR), rolling circle amplification (RCA), multiple
displacement
amplification (MDA), or random primer amplification (RPA). It is to be
understood that
amplification of the template polynucleotide chain prior to use is optional.
As such, the
template polynucleotide chain will not be amplified prior to use in some
examples.
Template/target polynucleotide chains can optionally be derived from synthetic
libraries.
Synthetic nucleic acids can have native DNA or RNA compositions or can be
analogs thereof.
[0127]
Biological samples from which the template polynucleotide chain can be
derived include, for example, those from a mammal, such as a rodent, mouse,
rat, rabbit, guinea
pig, ungulate, horse, sheep, pig, goat, cow, cat, dog, primate, human or non-
human primate; a
plant such as Arabidopsis thaliana, corn, sorghum, oat, wheat, rice, canola,
or soybean; an
algae such as Chlamydomonas reinhardtii; a nematode such as Caenorhabditis
elegans; an
insect such as Drosophila melanogaster, mosquito, fruit fly, honey bee or
spider; a fish such
as zebrafish; a reptile; an amphibian such as a frog or Xenopus laevis; a
Dictyostelium
discoideum; a fungi such as Pneumocy.slis carinii Takifugu rubnpes, yeast,
S'accharantoyees
cerevisiae or Schizosaccharomyces pombe; or a Plasmodi urn falciparum.
Template
polynucleotide chains 48 can also be derived from prokaryotes such as a
bacterium,
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Escherichia coli, staphylococci or Mycoplasma pneumoniae; an archa,ea; a virus
such as
Hepatitis C virus, Ebola virus or human immunodeficiency virus; or a viroid.
Template
polynucleotide chains can be derived from a homogeneous culture or population
of the above
organisms or alternatively from a collection of several different organisms,
for example, in a
community or ecosystem.
[01281 Moreover, template polynucleotide chains may not be
derived from natural
sources, but rather can be synthesized using known techniques. For example,
gene expression
probes or genotyping probes can be synthesized and used in the examples set
forth herein.
[0129] In some examples, template polynucleotide chains
can be obtained as
fragments of one or more larger nucleic acids. Fragmentation can be carried
out using any of
a variety of techniques known in the art including, for example, nebulization,
sonication,
chemical cleavage, enzymatic cleavage, or physical shearing. Fragmentation may
also result
from use of a particular amplification technique that produces amplicons by
copying only a
portion of a larger nucleic acid chain. For example, PCR amplification
produces fragments
having a size defined by the length of the nucleotide sequence on the original
template that is
between the locations where flanking primers hybridize during amplification.
The length of
the template polynucleotide chain may be in terms of the number of nucleotides
or in terms of
a metric length (e.g., nanomf..ters).
[0130] A population of template/target polynucleotide
chains, or amplicons
thereof, can have an average strand length that is desired or appropriate for
a particular
sequencing device. For example, the average strand length can be less than
about 100,000
nucleotides, about 50,000 nucleotides, about 10,000 nucleotides, about 5,000
nucleotides,
about 1,000 nucleotides, about 500 nucleotides, about 100 nucleotides, or
about 50 nucleotides.
Alternatively or additionally, the average strand length can be greater than
about 10
nucleotides, about 50 nucleotides, about 100 nucleotides, about 500
nucleotides, about 1,000
nucleotides, about 5,000 nucleotides, about 10,000 nucleotides, about 50,000
nucleotides, or
about 100,000 nucleotides. Alternatively or additionally, the average strand
length can be
greater than about 10 kilo nucleotides, about 50 kilo nucleotides, about 100
kilo nucleotides,
about 500 kilo nucleotides, about 1,000 kilo nucleotides, about 5,000 kilo
nucleotides, about
10,000 kilo nucleotides, about 50,000 kilo nucleotides, or about 100,000 kilo
nucleotides.
Alternatively or additionally, the average strand length can be greater than
about 10 mega
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nucleotides, about 50 mega nucleotides, about 100 mega nucleotides, about 500
mega
nucleotides, about 1,000 mega nucleotides, about 5,000 mega nucleotides, about
10,000 mega
nucleotides, about 50,000 mega nucleotides, or about 100,000 mega nucleotides.
The average
strand length for a population of target polynucleotide chains, or amplicons
thereof, can be in
a range between a maximum and minimum value set forth above.
[01311 In some cases, a population of template/target
polynucleotide chains can be
produced under conditions or otherwise configured to have a maximum length for
its members.
For example, the maximum length for the members can be less than about 100,000
nucleotides,
about 50,000 nucleotides, about 10,000 nucleotides, about 5,000 nucleotides,
about 1,000
nucleotides, about 500 nucleotides, about 100 nucleotides or about 50
nucleotides. For
example, the maximum length for the members can be less than about 100,000
kilo nucleotides,
about 50,000 kilo nucleotides, about 10,000 kilo nucleotides, about 5,000 kilo
nucleotides,
about 1,000 kilo nucleotides, about 500 kilo nucleotides, about 100 kilo
nucleotides or about
50 kilo nucleotides. For example, the maximum length for the members can be
less than about
100,000 mega nucleotides, about 50,000 mega nucleotides, about 10,000 mega
nucleotides,
about 5,000 mega nucleotides, about 1,000 mega nucleotides, about 500 mega
nucleotides,
about 100 mega nucleotides or about 50 mega nucleotides. Alternatively or
additionally, a
population of template polynucleotide chains, or amplicons thereof, can be
produced under
conditions or otherwise configured to have a minimum length for its members.
For example,
the minimum length for the members can be more than about 10 nucleotides,
about 50
nucleotides, about 100 nucleotides, about 500 nucleotides, about 1,000
nucleotides, about
5,000 nucleotides, about 10,000 nucleotides, about 50,000 nucleotides, or
about 100,000
nucleotides. For example, the minimum length for the members can be more than
about 10 kilo
nucleotides, about 50 kilo nucleotides, about 100 kilo nucleotides, about 500
kilo nucleotides,
about 1,000 kilo nucleotides, about 5,000 kilo nucleotides, about 10,000 kilo
nucleotides, about
50,000 kilo nucleotides, or about 100,000 kilo nucleotides. For example, the
minimum length
for the members can be more than about 10 mega nucleotides, about 50 mega
nucleotides,
about 100 mega nucleotides, about 500 mega nucleotides, about 1,000 mega
nucleotides, about
5,000 mega nucleotides, about 10,000 mega nucleotides, about 50,000 mega
nucleotides, or
about 100,000 inega nucleotides. The maximum and minimum strand length for
template
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polynucleotide chains in a population can be in a range between a maximum and
minimum
value set forth above.
[0132] As used herein, the term "signal" is intended to
mean an indicator that
represents information. Signals include, for example, an electrical signal and
an optical signal.
The term "electrical signal" refers to an indicator of an electrical quality
that represents
information. The indicator can be, for example, current, voltage, tunneling,
resistance,
potential, voltage, conductance, or a transverse electrical effect (and any
time-derivatives or
transients of theses). An "electronic current" or "electric current" refers to
a flow of electric
charge. In an example, an electrical signal may be an electric current passing
through a
nanopore, and the electric current may flow when an electric potential
difference is applied
across the nanopore.
[0133] The term "substrate" refers to a rigid, solid
support that is insoluble in
aqueous liquid and is incapable of passing a liquid absent an aperture, port,
or other like liquid
conduit. In the examples disclosed herein, the substrate may have wells or
chambers defined
therein. Examples of suitable substrates include glass and modified or
functionalized glass,
plastics (including acrylics, polystyrene and copolymers of styrene and other
materials,
polypropylene, polyethylene, polybut3rIene, polyurethanes, polytetrafl
uoroethy ene (PTF E)
(such as TEFLON from Chemours), cyclic olefins/cyclo-olefin polymers (COP)
(such as
ZEONOR from Zeon), polyimides, etc.), nylon, ceramics, silica or silica-based
materials,
silicon and modified silicon, carbon, metals, inorganic glasses, and optical
fiber bundles.
[0134] As used herein, the term "interstitial region"
refers to an area in a
substrate/solid support or a membrane, or an area on a surface that separates
other areas,
regions, features associated with the support or membrane or surface. For
example, an
interstitial region of a membrane can separate one nanopore of an array from
another nanopore
of the array. For another example, an interstitial region of a substrate can
separate one trans/cis
well from another trans/cis well. The two areas that are separated from each
other can be
discrete, i.e., lacking physical contact with each other. In many examples,
the interstitial region
is continuous whereas the areas are discrete, for example, as is the case for
a plurality of
nanopores defined in an otherwise continuous membrane, or for a plurality of
wells defined in
an otherwise continuous substrate/support. The separation provided by an
interstitial region
can be partial or full separation. Interstitial regions may have a surface
material that differs
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from the surface material of the features defined in the surface. For example,
the surface
material at the interstitial regions may be a lipid material, and a nanopore
formed in the lipid
material can have an amount or concentration of polypeptide that exceeds the
amount or
concentration present at the interstitial regions. In some examples, the
polypeptide may not be
present at the interstitial regions.
01351 The terms top, bottom, lower, upper, on, etc. are
used herein to describe the
device/nanopore sequencer and/or the various components of the device. It is
to be understood
that these directional terms are not meant to imply a specific orientation,
but are used to
designate relative orientation between components. The use of directional
terms should not be
interpreted to limit the examples disclosed herein to any specific
orientation(s). As used herein,
the terms "upper", "lower", "vertical", "horizontal" and the like are meant to
indicate relative
orientation.
[0136] As used herein, "cis" refers to the side of a
nanopore opening through which
an analyte or modified analyte enters the opening or across the face of which
the analyte or
modified analyte moves.
[0137] As used herein, "trans" refers to the side of a
nanopore opening through
which an analyte or modified analyte (or fragments thereof) exits the opening
or across the
face of which the analyte or modified analyte does not move.
[0138] As used herein, by "translocation," it is meant
that an analyte (e.g., DNA)
enters one side of an opening of a nanopore and move to and out of the other
side of the
opening. It is contemplated that any embodiment herein comprising
translocation may refer to
electrophoretic translocation or non-electrophoretic translocation, unless
specifically noted.
An electric field may move an analyte (e.g., a polynucleotide) or modified
anal yte. By
"interacts," it is meant that the analyte (e.g., DNA) or modified analyte
moves into and,
optionally, through the opening, where "through the opening" (or
"translocates") means to
enter one side of the opening and move to and out of the other side of the
opening. Optionally,
methods that do not employ electrophoretic translocation are contemplated. In
some
embodiments, physical pressure causes a modified analyte to interact with,
enter, or translocate
(after alteration) through the opening. In some embodiments, a magnetic bead
is attached to an
analyte or modified analyte on the trans side, and magnetic force causes the
modified analyte
to interact with, enter, or translocate (after alteration) through the
opening. Other methods for
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translocation include but not limited to gravity, osmotic forces, temperature,
and other physical
forces such as centripetal force.
[0139] As used herein, the terms "well", "cavity",
"reservoir" and "chamber" are
used synonymously, and refer to a discrete feature defined in the device that
can contain a fluid
(e.g., liquid, gel, gas). A cis well is a chamber that contains or is
partially defined by a cis
electrode, and is also fluidically connected to a middle well where
measurements occur (for
example, by a FET, or by a metal electrode connected to an amplifier, a data
acquisition device,
or other signal conditioning elements such as analog filters, buffers, gain
amplifiers, ADCs,
etc.). The middle well in turn is fluidically connected to a trans
well/chamber, in some
examples. Examples of an array of the present device may have one cis well,
for example one
global cis chamber/reservoir, or multiple cis wells. The trans well is a
single chamber that
contains or is partially defined by its own trans electrode, and is also
fluidically connected to
a cis well. In examples including multiple trans wells, each trans well is
electrically isolated
from each other trans well. Further, it is to be understood that the cross-
section of a well taken
parallel to a surface of a substrate at least partially defining the well can
be curved, square,
polygonal, hyperbolic, conical, angular, etc. As used herein, "field-effect
transistors" or
"FETs" typically include doped source/drain regions that are formed of a
semiconductor
material, e.g., silicon, germanium, gallium arsenide, silicon carbide, etc.,
and are separated by
a channel region. A n-FET is a FET having an n-channel in which the current
carriers are
electrons. A p-FET is a FET having a p-channel in which the current carriers
are holes.
Source/drain regions of a n-FET device may include a different material than
source/drain
regions of a p-FET device. In some examples, the source/drain regions or the
channel may not
be doped. Doped regions may be formed by adding dopant atoms to an intrinsic
semiconductor.
This changes the electron and bole carrier concentrations of the intrinsic
semiconductor at
thermal equilibrium. A doped region may be p-type or n-type. As used herein,
"p-type" refers
to the addition of impurities to an intrinsic semiconductor that creates a
deficiency of valence
electrons. For silicon, example p-type dopants, i.e., impurities, include but
are not limited to
boron, aluminum, gallium, and indium. As used herein, "n-type" refers to the
addition of
impurities that contribute free electrons to an intrinsic semiconductor. For
silicon, example n-
type dopants, i.e., impurities, include but are not limited to, antimony,
arsenic, and phosphorus.
The dopant(s) may be introduced by ion implantation or plasma doping.
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[0140]
For example, in an integrated circuit having a plurality of metal oxide
semiconductor field effect transistors (MOSFETs), each MOSFET has a source and
a drain
that are formed in an active region of a semiconductor layer by implanting n-
type or p-type
impurities in the layer of semiconductor material. Disposed between the source
and the drain
is a channel (or body) region. Disposed above the body region is a gate
electrode. The gate
electrode and the body are spaced apart by a gate dielectric (gate oxide)
layer. The channel
region connects the source and the drain, and electrical current flows through
the channel
region from the source to the drain. The electrical current flow is induced in
the channel region
by a voltage applied at the gate electrode.
[0141]
In some embodiments, the channel of a FET sensor located between the
source and drain may be covered by a relatively thin layer of a gate oxide,
for example a
thermally grown silicon dioxide layer. Alternatively, a thin layer of an
insulator may be formed
of h
dielectrics, such as }1102, Al2O3, silicon nitroxides, S i3N4, TiO2,
Ta205, Y203, T A203,
ZrO2, ZrSiO4, barium strontium titanate, lead zirconate titanate, ZrSix0y, or
ZrA1,0y. The layer
of gate oxide may be about 10 rim in thickness, or in other examples, less
than about 9, about
8, about 7, about 6, about 5, about 4, about 3, about 2, or about I rim in
thickness.
[0142]
Non-planar transistor device architectures, such as nanosheet (or
nanowire)
transistors, can provide increased device density and increased performance
over planar
transistors. A -gate-all-around" transistor is a transistor in which the gate
is structured to wrap
around the channel. A "nanosheet transistor" refers to a type of FET that may
include a
plurality of stacked nanosheets extending between a pair of source/drain
regions, forming a
channel. Nanosheet transistors, in contrast to conventional planar FETs, may
include a gate
stack that wraps around the full perimeter of multiple nanosheet channel
regions. Nanosheet
transistor configurations enable fuller depletion in the nanosheet channel
regions and reduce
short-channel effects. "Nanowire transistors" may be similar to nanosheet
transistors, except
the channel may include nanowires instead of nanosheets. The gate-all-around
structure in
nanosheet or nanowire transistors can provide very small devices with better
switching control,
lower leakage current, faster operations, and lower output resistance.
[01431
A way of increasing channel conductivity and decreasing FET size is to
form the channel as a nanostructure. For example, a gate-all-around (GAA)
nanosheet FET is
an architecture for providing a relatively small FET footprint by forming the
channel region as
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a series of nanosheets. In a GAA configuration, a nanosheet-based FET includes
a source
region, a drain region and stacked nanosheet channels between the source and
drain regions.
A gate surrounds the stacked nanosheet channels and regulates electron flow
through the
nanosheet channels between the source and drain regions. GAA nanosheet FETs
may be
fabricated by forming alternating layers of channel nanosheets and sacrificial
nanosheets. The
sacrificial nanosheets are released from the channel nanosheets before the FET
device is
finalized. For n-type FETs, the channel nanosheets are typically silicon (Si)
and the sacrificial
nanosheets are typically silicon germanium (SiGe). For p-type FETs, the
channel nanosheets
are typically. SiGe and the sacrificial nanosheets are typically Si. In some
implementations, the
channel nanosheet of a p-FET can be SiGe or Si, and the sacrificial nanosheets
can be Si or
SiGe. Forming the GAA nanosheets from alternating layers of channel nanosheets
formed from
a first type of semiconductor material (e.g., Si for n-type FETs, and SiGe for
p-type FETs) and
sacrificial nanosheets formed from a second type of semiconductor material
(e.g., SiGe for n-
type FETs, and Si for p-type FETs) provides superior channel electrostatics
control, which is
beneficial for continuously scaling gate lengths down to seven nanometer CMOS
technology
and below. The use of multiple layered SiGe/Si sacrificial/channel nanosheets
(or Si/SiGe
sacrificial/channel nanosheets) to form the channel regions in GAA FET
semiconductor
devices provides desirable device characteristics, including the introduction
of strain at the
interface between SiGe and Si.
[0144] In some examples, a "nanowire" is characterized by
a critical dimension of
less than about 30 nm, while a "nanosheet" is characterized by a critical
dimension of about
30 nm or greater. In exemplary devices, the critical dimension is measured
along the gate. In
that direction, if the width of the channel is small, the channel cross-
section is like a "wire"
whereas if the width of the channel is large, the channel cross-section is
like a "sheet."
[0145] In some examples, the smallest dimension of the
nanosheet or nanowire is
between about 1-10, about 1-50, about 1-100, about 1-500, or about 1-1000 nm.
In some
examples, the smallest dimension of the nanosheet or nanowire is between about
1-5, about 3-
10, about 5-15, about 10-20, about 15-30, about 20-40, about 30-50, about 40-
75, about 50-
100, about 75-150, about 100-200, about 150-300, about 200-400, about 300-500,
about 400-
750, or about 500-1000 nm. In some examples, the smallest dimension of the
nanosheet is at
least about 3, about 5, about 7, about 10, about 15, about 20, about 50, about
100, about 150,
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about 200, about 250, about 300, about 350, about 400, about 450, about 500,
about 600, about
700, about 800, about 900, about 1000, about 2000, about 2500, about 3000,
about 4000, or
about 5000 times smaller than the other two dimensions of the nanosheet. In
some examples,
the smallest dimension of the nanosheet is between about 2-5, about 3-7, about
5-10, about 7-
15, about 10-20, about 15-50, about 20-100, about 50-150, about 100-200, about
150-250,
about 200-300, about 250-350, about 300-400, about 350-450, about 400-500,
about 450-600,
about 00-700, about 600-800, about 700-900, about 800-1000, about 900-2000,
about 1000-
2500, about 2000-3000, about 2500-4000, or about 3000-5000 times smaller than
the other two
dimensions of the nanosheet. In some examples, the smallest dimension of the
nanosheet is at
most about 3, about 5, about 7, about 10, about 15, about 20, about 50, about
100, about 150,
about 200, about 250, about 300, about 350, about 400, about 450, about 500,
about 600, about
700, about 800, about 900, about 1000, about 2000, about 2500, about 3000,
about 4000, or
about 5000 times smaller than the other two dimensions of the nanosheet. In
some examples,
the biggest dimension of the nanowire is at least about 3, about 5, about 7,
about 10, about 15,
about 20, about 50, about 100, about 150, about 200, about 250, about 300,
about 350, about
400, about 450, about 500. about 600, about 700, about 800, about 900, about
1000, about
2000, about 2500, about 3000, about 4000, or about 5000 times bigger than the
other two
dimensions of the nanowire. In some examples, the biggest dimension of the
nanowire is
between about 2-5, about 3-7, about 5-1.0, about 7-15, about 10-20, about 15-
50, about 20-100,
about 50-150, about 100-200, about 150-250, about 200-300, about 250-350,
about 300-400,
about 350-450, about 400-500, about 450-600, about 500-700, about 600-800,
about 700-900,
about 800-1000, about 900-2000, about 1000-2500, about 2000-3000, about 2500-
4000, or
about 3000-5000 times bigger than the other two dimensions of the nanowire. In
some
examples, the biggest dimension of the nanowire is at most about 3, about 5,
about 7, about
10, about 15, about 20, about 50, about 100, about 150, about 200, about 250,
about 300, about
350, about 400, about 450, about 500, about 600, about 700, about 800, about
900, about 1000,
about 2000, about 2500, about 3000, about 4000, or about 5000 times bigger
than the other
two dimensions of the nanowire.
[01461 The aspects and examples set forth herein and
recited in the claims can be
understood in view of the above definitions.
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Overview
101471 In some aspects, the disclosure provides a process
integration scheme for
achieving a horizontal architecture of a nanopore sequencing device, a
nanopore sequencing
device having a horizontal architecture, and a method of using such a device.
[0148] Disclosed herein is a nanopore sequencing device
includes a middle well
associated with a sensing electrode, a cis well associated with a cis
electrode, and a trans well
associated with a trans electrode. In some embodiments, the middle well is
positioned between
the cis well and the trans well, and the cis well, middle well, and the trans
well are oriented
side-by-side horizontally. In some embodiments, the cis well is oriented
vertically with respect
to the middle and/or trans well. In some embodiments, the trans well is
oriented vertically with
respect to the middle well. In some embodiments, the device may comprise one
or more
common cis well and one or more common trans well that are shared by all
sequencing unit
cells For example, a common trans wall and a common cis well may be in fluid
communication
with a plurality of middle wells. In some embodiments, the cis and trans wells
may be
considerably larger than each of the middle wells to avoid ion depletion,
while each middle
well may contain its own, individually addressable sensing electrode.
10149.1 The nanopore sequencing device further includes a
first nanoscale opening,
e.g., a nanoscale opening arranged in a nanopore, disposed between the cis
well and the middle
well, and a second nanoscale opening, e.g., a horizontal nanochannel, formed
on the surface of
the substrate between the trans well and the middle well. In some embodiments,
the
nanochannel is fabricated horizontally on the surface of the substrate. For
example, in some
embodiments, the nanochannel is formed by etching a semiconductor wafer. In
some
embodiments, the nanochannel is formed by patterned layers over a
semiconductor wafer. In
some embodiments, the nanochannel does not comprise a through-hole in the
substrate. The
middle well of the nanopore sequencing device fluidically connects the cis
well to the trans
well.
[0150] When one or more nucleotides of a target DNA are
near or at the first
nanoscale opening, e.g., near or at the nanopore, the electrical resistance of
the first nanoscale
opening may vary in response to the identity of the one or more nucleotides.
The second
nanoscale opening, e.g., the nanochannel, may have a fixed, or substantially
fixed electrical
resistance. In some embodiments, the length of the nanochannel is chosen for
its specific
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electrical resistance. In some embodiments, the electrical resistance of the
first nanochannel is
altered by changing the length of the nanochannel. In some embodiments, such a
device may
further include electronics to actively control the second nanoscale opening,
e.g., to control the
nanochannel. For example, pressure pulse generators, air/gas bubble
generators/annihilators,
stimuli-responsive polymers or gels may be used to control
liquid/ionic/electric flow in the
second nanoscale opening. In some embodiments, the electronics or actuators
may be formed
under (or around) the second nanoscale opening, e.g., formed under (or around)
the
nanochannel.
[0151] In some embodiments, the device may further include
one or more
additional middle wells, each additional middle well associated with a
respective additional
sensing electrode, where a respective additional first nanoscale opening is
disposed between
the cis well and each additional middle well, where a respective additional
second nanoscale
opening is disposed between the trans well and each additional middle well,
and where the one
or more additional middle wells fluidically connect the cis well to the trans
well. In some
embodiments, at least some of the additional first nanoscale openings may be
arranged in
nanopores. In some embodiments, at least some of the additional second
nanoscale openings
may be arranged in nanochannels. In some embodiments, an array of middle wells
is formed
on a substrate, the middle wells are in fluid communication with one or more
common trans
well or trans channel, and also in fluid communication with one or more common
cis well or
cis channel.
[0152] In some embodiments, to use such a device for
sequencing a biopolymer, a
method may include introducing an electrolyte into the cis well, the trans
well, and at least one
of the middle wells. The method may further include applying a voltage between
the cis
electrode and the trans electrode to control the motion of the biopolymer. The
method may
further include measuring, from the respective sensing electrode, an electric
potential of the
electrolyte in the middle well, where an electrical resistance of the
respective first nanoscale
opening, e.g., nanopore, varies in response to an identity of one or more
monomers in the
biopolymer, the one or more monomers being near or at the respective first
nanoscale opening.
[01531 In some embodiments, a method of manufacturing such
a device may
include forming a bottom wafer comprising at least one of the second nanoscale
openings, e.g.,
nanochannels, at least one of the respective sensing electrodes, and a first
patterned layer. In
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some embodiments, the nanochannels are horizontal nanochannels. In some
embodiments, the
nanochannels are fabricated into the substrate. For example, in some
embodiments, the
nanochannels are etched into a semiconductor wafer. In some embodiments, the
nanochannels
are formed by patterned layers over a semiconductor wafer. The method may
further include
forming a top wafer comprising a second patterned layer. The method may
further include
aligning the first patterned layer with the second patterned layer. The method
may further
include bonding the first patterned layer with the second patterned layer at a
plurality of
locations via an adhesive, such that the cis well, the trans well, and at
least one of the respective
middle wells are formed between the bottom wafer and the top wafer.
[01541 In certain embodiments, a nanopore sequencing
device having a horizontal
structure includes one or a combinations of the following aspects:
[0155] (i) Ease of or more precise manufacturing process
eliminating the need to
(a) etch high aspect ratio through-silicon vias/caviti es into the Si
substrate, (b) perform
backside wafer processing that may compromise the wafer front-side, (c) use
expensive 193
rim lithography masks, and (d) perform wafer-to-wafer bonding. Fabricating
horizontal
nanochannels of specific fluidic/electric resistance reduces the number of
undesirable steps
(such as complex multiple etch steps, deposition of sacrificial etch stop
layers, re-oxidation
and wafer backside processing steps) compared to vertical through-Si
nanochannels.
[0156] (ii) In terms of manufacturability and
reproducibility, the uniformity of
horizontal nanochannel width (or diameter), both along a single nanochannel
and across
nanochannels on a wafer, can be better controlled compared to a vertical
implementation of
nanochannels. Moreover, non-destructive metrology can be employed to assess
uniformity of
critical dimensions across a nanochannel and wafer.
[0157] (iii) Ability to increase nanochannel resistance by
increasing overall
nanochannel length, e.g., using a meandering/tortuous/serpentine layout as
shown in FIG. 3B
instead of a linear layout. Variations of the nanochannel resistance can be
implemented easily
on the same substrate by changing the nanochannel length. Unlike the length of
vertical
nanochannels in a vertical device implementation, the length of horizontal
nanochannels is not
limited by the substrate thickness and multiple lengths/resistances can be
achieved on one
wafer thus allowing faster learning cycles and process optimization. A wider
nanochannel
width (or diameter) is beneficial for reducing the pressure inside the middle
well, but this wider
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nanochannel width may need to be accompanied by an increased nanochannel
length to achieve
the required nanochannel resistance, which poses difficulties for a vertical
device
implementation relying on through-Si etch.
[01581 (iv) The ability to achieve longer nanochannels
relaxes the nanochannel
diameter and eliminates the need for subsequent deposition of thick (e.g., >
500 nm) layers to
reduce the nanochannel width. For example, one could start with a nanochannel
width of 350
nm defined using inexpensive i-line lithography or nanoimprint lithography and
deposit a 135
nm oxide or nitride layer to achieve a final nanochannel width of 80 nm, as
shown in FIG. 5F'.
[0159] (v) Ability to integrate active electronics
underneath the nanochannel to
control resistance and/or act as an electronic switch/valve. Implementation of
horizontal
nanochannels also allows integration of active circuitry/actuators underneath
or in the vicinity
of each nanochannel to control/regulate the nanochannel resistance or other
behaviors. For
example, an integrated heating element (e.g., resistor) could be used to
generate nanobubbles
(water vapor) that block a nanochannel and thus switch off the
current/ionic/fluidic flow
through that respective nanochannel and sequencing unit cell. This helps avoid
current/ionic/fluidic flow through sequencing unit cells which are identified
as corrupted or
non-functional. For another example, a piezoelectric element (e.g., ultrasonic
actuator) could
be used to eliminate bubbles or other unwanted debris (e.g., clogged DNA
template) in the
nanochannel. Other control modalities employed in digital microfluidics (such
as
electrowetting) or valve elements involving stimuli-responsive polymers or
hydrogels may
also be integrated into the nanochannel of the disclosed device.
[0160] (vi) Improved device robustness by integration of
cis and trans fluidic wells
onto the chip thus eliminating the need for complex setup fixtures or hardware
integration.
[0161] (vii) The cis and trans wells can be integrated
directly onto the chip by
means of wafer-to-wafer bonding, thus creating a fully contained flowcell
that, in some
embodiments, only requires inlet/outlet ports for fluidic and electrical
connections to an
external fixture/cartridge (see FIG. IA for an example). External fluidic
reservoirs and
electrodes can be made large enough to maintain constant ion concentrations
inside the
flowcell without risk of ion depletion.
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Example Nanopore Sequencing Devices
101621 FIG. IA is a cross-sectional side view of an
example nanopore sequencing
device 110 having a horizontal architecture. Shown is a schematic illustration
of two
sequencing unit cells laid out side-by-side and axisymmetric with respect to
the trans well, i.e.,
the two sequencing unit cells are sharing the same trans well. A. flowcell 170
of the nanopore
sequencing device 110 is arranged between a bottom wafer 157 and a top wafer
167. The
flowcell 170 may be filled with an electrolyte. Above the top wafer 167, a top
portion 180 of
the nanopore sequencing device 110 may be connected to external fluidic
fixtures. Openings
or holes 160 may be formed within the top wafer 167 to allow fluidic
communications.
[0163] The nanopore sequencing device 110 includes a cis
electrode 130 associated
with a cis well 114. The nanopore sequencing device 110 further includes a
trans electrode 134
associated with a trans well 116. In one example, the cis electrode 130 and
the trans electrode
134 are arranged in an at least substantially horizontal direction with
respect to the wafers. In
other examples, the cis electrode and the trans electrode may be in any
suitable orientation
relative to each other and to the wafers. A divider 139 may separate the cis
electrode 130/cis
well 114 from the trans electrode 134/trans well 116. A middle well 115 is
positioned between
the cis well 114 and the trans well 116, and the cis well, middle well, and
the trans well are
oriented side by side horizontally.
[0164] The cis well 114 is connected to a nanopore 123, in
which a first nanoscale
opening is formed. In some embodiments, the nanopore 123 may be formed in a
protein 118
disposed into a membrane 124. In some embodiments, the membrane 124 may be
arranged
vertically with respect to the wafers and the nanochannel, on a side of the
cis well 114. The
nanopore 123 provides a fluidic pathway for an electrolyte to pass between the
cis well 114
and the middle well 115. The nanopore 123 fluidically communicates with a
nanochannel 125
through the middle well 115. The nanochannel 125, in which a second nanoscale
opening is
formed, provides a fluidic/ionic/electric pathway for the electrolyte/current
to pass between
the middle well 115 and the trans well 116. The trans electrode 134 may be
operably connected
to a voltage supplier 111. The flowcell 170 includes the cis well 114, the
trans well 116, a
plurality of middle wells and their respective nanopores and nanochannels, all
of which are in
fluidic communication. A characteristic width of the middle well 115 may be
about 5 lam, 10
gm, 20 gm, 30 pm, 40 p.m, 50 gm, 60 p.m, 70 lam, 80 gm, 90 gm, 100 gm, 150 gm,
200 gm, or
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any value therebetween. . A characteristic depth of the middle well 115 may be
about 5 pm,
gm, 20 p.m, 30 gm, 40 gm, 50 gm, 60 gm, 70 gm, 80 gm, 90 pm, 100 gm, 1501AM,
200 gm,
or any value therebetween. A characteristic width of the cis well or the trans
well may be about
10 gm, 50 gm, 100 gm, 200 gm, 300 gm, 400 gm, 500 gm, 600 gm, 700 gm, 800 gm,
900
gm, 1 mm, 5 mm, 10 mm, or any value therebetween. The walls of the middle well
115 may
be at least partly defined by wall structures 147, 159 and 149.
[0165] In some embodiments, the nanochannel 125 may be
formed on the surface
of the wafer horizontally or at least partially horizontally with respect to
the wafers. In some
embodiments, the nanochannel does not comprise a through-hole in the
substrate. The width
or diameter of the nanochannel 125 may be about 5 nrn, 10 nm, 15 mil, 20 nm,
30 nm, 40 nm,
50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 150 nu', 200 nm, or any value
therebetween.
The width of the nanochannel 125 may be adjusted by a deposited layer 137. The
width of the
nanochannel may be narrowed by the deposited layer by about 5%, 10%, 15%, 20%,
25%,
30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or any value
therebetween. In some embodiments, the nanochannel 125 may have a meandering,
serpentine
or tortuous path, in order to achieve a longer nanochannel path length while
keeping the
footprint small. A characteristic size of the nanochannel footprint (e.g., the
length of the
footprint) may be about 5 gm, 10 gm, 15 gm, 20 gm., 25 gm, 30 pm, 40 gm, 50
gm, 100 gm,
200 pm, 300 gm, 400 pm, 500 gm, or any value therebetween. A total. path
length of a
nanochannel may be about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35,
40, 45, 50 times, or
any value therebetween, of the characteristic size of the nanochannel
footprint. A longer
nanochannel path length may allow for a larger nanochannel resistance. The
tortuous path may
be a rectangular wave shape, a sine wave shape, a sawtooth shape, a zigzag
shape, a spiral
shape, or any combination thereof. The tortuous path may include a rectangular
wave shape, a
sine wave shape, a sawtooth shape, a zigzag shape, a spiral shape, or any
combination thereof
as a part of its shape.
[01661 In some embodiments, a nanopore sequencing device,
such as the device
110, may further include electronics or actuators arranged relatively
underneath, above, to the
side(s) of, and/or around some or all of the nanochannels to actively control
liquid flow in
some or all of the nanochannels. In some examples, micro-heaters (such as a
resistive heater
illustrate in FIG. 7A), optical transducers, pressure-based transducers,
electromagnetic
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acoustic transducers may be included. For another example, ultrasound
transducers may be
used to generate pressure pulses in the liquid to eliminate bubbles or other
unwanted debris
from the nanochannels. Further methods of manipulating liquid flows can be
found in Arango,
Yulieth, et al. -Electro-actuated valves and self-vented channels enable
programmable flow
control and monitoring in capillary-driven microfluidics." Science Advances
6.16 (2020):
eaay8305, the disclosure of which is incorporated herein by reference.
[01671
In some embodiments, a nanopore sequencing device may further include
electrodes to generate gas bubbles by electrolysis of the fluid in the
nanochannels to block the
liquid flow. For example, in the event of rupture and/or fai lure of the
membrane and/or
nanopore, gas bubbles can be generated to block ionic current flow of the non-
performing
sequencing unit cell so that the other performing cis/trans cells may continue
to properly
perform. The electrodes can create an electric field across the nanochannel
and/or providing
electron source/sink for electrolysis of the fluid within the electric field
[01681
In some embodiments, the first and the second electrodes can be above
and/or below the nanochannel. In some embodiments, the first and the second
electrodes can
be on each of the sides of the nanochannel. In some embodiments, the first
electrode can be
surrounding a first portion of the channel and the second electrode can be
surrounding a second
portion of the channel. In one example shown in FIG. 1A., the pair of
electrodes 1001 and
1002 may be formed underneath the nanochannel 125. In another example shown in
FIG. 8,
the pair of electrodes 1001 and 1002 may be formed at outside of the
nanochannel 125, at about
the entrance and the exit of the nanochannel 125. FIG. I B illustrates another
example for
generating water electrolysis. As shown in this figure, in some embodiments, a
bubble can be
generated on the bottom sensing elec-trode/FET as one of the electrode pair,
and the CIS
electrode and/or Trans electrode or another electrode can be utilized as the
other electrode pair.
[0169]
An electric field is created between the first and the second electrodes
by
having the electrodes at different potentials. For example, the voltage
applied on the first
electrode may range from +1 Volt to -1-2 Volt and the voltage applied on the
second electrode
may range from -1 Volt to -2 Volt, or vice versa. In some embodiments, one of
the electrodes
can be biased and other electrode can be grounded. In some embodiments, one of
the
electrodes can be biased and other electrode can be floating. In some
embodiments, both of
the electrodes can be biased but at different potentials. In some embodiments,
the electrodes
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for generating water electrolysis may be non-reactive with the electrolyte.
For example, the
electrodes may be formed of platinum, iridium, ruthenium, palladium, tantalum,
gold, TiN, or
any combination thereof.
[01701 As exemplified in FIG. IA, a sensing electrode 122
may be arranged in the
bottom wafer (or the first substrate) 157 and may be exposed to the
electrolyte in the middle
well 115. The sensing electrode 122 may be used to detect the electrical
potential of the
electrolyte in the middle well and to transmit the detected signal to a
voltage detector circuit
or a field effect transistor. The voltage detector circuit or the field effect
transistor may be
external to the nanopore sequencing device 110, or may be arranged in the
bottom wafer 157.
The sensing electrode 122 may be made of corrosion-resistant metals with
respect to the
electrolyte. The sensing electrode 122 may be made of platinum, iridium,
ruthenium,
palladium, tantalum, gold, TiN, or any combination thereof. No electrochemical
reaction may
occur at the sensing electrode 122.
[01711 The membrane in a nanopore sequencing device may be
formed. from any
suitable natural or synthetic material. In some embodiments, the membrane may
be formed of
a non-permeable or semi-permeable material. In an example shown in FIG. I A,
the membrane
124 is selected from the group consisting of a lipid and a biomimetic
equivalent of a lipid. The
nanopore in a nanopore sequencing device may be any of the biological
nanopores, solid-state
nanopores, hybrid nanopores, and synthetic nanopores described herein. I some
embodiments,
the nanopore may be a hollow defined by, for example: a polynucleotide
structure, a
polypeptide structure, or a solid-state structure, e.g., a carbon nanotube,
which is disposed in
the membrane. In some embodiments, the membrane may be a synthetic membrane
(e.g., a
solid-state membrane, one example of which is silicon nitride), and the
nanopore is in a hollow
extending through the membrane. In an example, the nanopore inner diameter
ranges from
about 0.5 nm to about 3 nm. In another example, the nanopore inner diameter
ranges from
about 1 nm to about 2 nm. In yet another example, the nanopore inner diameter
ranges from
about 1 nm to about 3 nm. The example ranges for the nanopore given above are
intended to
be the smallest diameter of the nanopore.
[01721 For example, as shown in FIG. 1A, a protein 118
having a hollow may be
inserted into the membrane 124 directly, or the membrane may be formed around
the protein.
In an example, the protein may insert itself into a formed lipid bilayer
membrane. For example,
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a protein in its monomeric form or polymeric form (e.g., an octamer) may
insert itself into the
lipid bilayer and assemble into a transmembrane pore. In another example, the
protein may be
added to a grounded side of a lipid bilayer at a desirable concentration where
it will insert itself
into the lipid bilayer. In still another example, the lipid bilayer may be
formed across an
aperture in a polytetrafluoroethylene (PTFE) film or any photo-patternable
material (e.g., NIL
resin), polyimide, silicon, or glass that is chemically stable and does not
dissolve in the
chemicals used for sequencing, and positioned between the cis well and the
middle well. The
protein may be added to the cis compartment, and may insert itself into the
lipid bilayer at the
area where the FITE aperture is formed. In yet a further example, the protein
may be tethered
to a solid support (e.g., silicon, silicon oxide, quartz, indium tin oxide,
gold, polymer, etc.). A
tethering molecule, which may be part of the protein itself or may be attached
to the protein,
may attach the protein to the solid support. The attachment via the tethering
molecule may be
such that a single protein is immobilized between the cis well and the middle
well A lipid
bilayer may then be formed around the protein.
[0173] The cis electrode that is used for nanopore
sequencing devices as disclosed
herein depends, at least in part, upon the redox couple in the electrolyte. As
examples, the cis
electrode may be gold (Au), platinum (Pt). carbon (C) (e.g., graphite,
diamond, etc.), palladium
(Pd), silver (Ag), copper (Cu), or the like. In an example, the cis electrode
may be a
silver/silver chloride (Ag/A.gCI) electrode. In one example, the cis well is
capable of
maintaining the electrolyte in contact with the first nanoscale opening. In
some examples, the
cis well may be in contact with an array of nanopores, and thus is capable of
maintaining the
electrolyte in contact with each of the nanopores in the array.
[0174] The trans electrode that is used for nanopore
sequencing devices as
disclosed herein depends, at least in part, upon the redox couple in the
electrolyte. As
examples, the trans electrode may he gold (Au), platinum (Pt), carbon (C)
(e.g., graphite,
diamond, etc.), palladium (Pd), silver (Ag), copper (Cu), or the like. In an
example, the trans
electrode may be a silver/silver chloride (AglAgC1) electrode.
[0175] In some examples, the relevant electrochemical half-
reactions at the
electrodes for a Ag/AgCI electrode in NaC1 or KCI solution, are:
Cis (cathode): AgC1+ e 4 Ag 4- Cl, and
Trans (anode): Ag Cl- 4 AgC1 .
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[0176] For every unit charge of current, one Cl atom is
consumed at the trans
electrode. Though the discussion above is in terms of an AglAgC1 electrode in
NaCI or KC1
solution, it is to be understood that any electrode/electrolyte pair that may
be used to pass the
current may apply.
[0177] In use, an electrolyte may be filled into the cis
well, the middle well, and
the trans well. In alternative examples, the electrolyte in the cis well, the
middle well, and the
trans well may be different. The electrolyte may be any electrolyte that is
capable of
dissociating into counter ions (a cation and its associated anion). As
examples, the electrolyte
may be an electrolyte that is capable of dissociating into a potassium cation
(K+) or a sodium
cation (Na'). This type of electrolyte includes a potassium cation and an
associated anion, or
a sodium cation and an associated anion, or combinations thereof. Examples of
potassium-
containing electrolytes include potassium chloride (KCl), potassium
ferricyanide
(K3[Fe(CN)6] = 3H20 or .K4Fe(CN)61 = 3H20), or other potassium-containing
electrolytes (e.g.,
bicarbonate (KHCO3) or phosphates (e.g., KH2PO4, K2HPO4, K3PO4). Examples of
sodium-
containing electrolytes include sodium chloride (NaC1) or other sodium-
containing
electrolytes, such as sodium bicarbonate (NaHCO3), sodium phosphates (e.g.,
NaH2PO4,
Na2HPO4 or NaR04). As another example, the electrolyte may be any electrolyte
that is
capable of dissociating into a ruthenium-containing cation (e.g., ruthenium
hexamine, such as
[Ru(ICH3)6]2+ or [Ru(NH3)6]3'). Electrolytes that are capable of dissociating
into a lithium
cation (Li'), a rubidium cation (R134), a magnesium cation No, or a calcium
cation (Ca')
may also be used.
[0178] FIG. 2 shows an equivalent circuit diagram 210 of a
na.nopore sequencing
device described herein. To use the nanopore sequencing device, an electrolyte
is introduced
into each of the cis well, the trans well, and the middle well. A voltage
difference V is applied
between the cis electrode (indicated as node 230 in FIG. 2) and the trans
electrode (indicated
as node 234 in FIG. 2) by the voltage supplier (indicated as feature 111 in
FIG. IA and feature
211 in FIG. 2). During operation, the range of applied voltages can be
selected from about -
0.1 mV to upwards of about 0.1 mV, from about -0.5 mV to upwards of about 0.5
mV, from
about -1 mV to upwards of about 1 mV, from about -1.5 mV to upwards of about
1.5 mV, from
about -2.0 mV to upwards of about 2.0 mV, from about -3.0 mV to upwards of
about 3.0 mV,
from about -5.0 mV to upwards of about 5.0 mV, from about -0.1 V to upwards of
about 0.1
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V, from about -0.5 V to upwards of about 0.5 V, from about -1 V to upwards of
about 1 V,
from about -1.5 V to upwards of about 1.5 V. from about -2.0 V to upwards of
about 2.0 V.
from about -3.0 V to upwards of about 3.0 V, or from about -5.0 V to upwards
of about 5.0 V.
In some instances, the voltage polarity may be applied such that a negatively
charged nucleic
acid is electrophoretically driven towards the trans electrode. In some
instances, the voltage
polarity may be applied such that a positively charge protein is
electrophoretically driven
towards the trans electrode. In some instances, the voltage can be reduced, or
the polarity
reversed, to facilitate appropriate function of the device.
[0179] In some examples, a polynucleotide is driven
through the nanopore. During
a nanopore sequencing operation, the application of the voltage difference V
across the cis
electrode 130 and the trans electrode may force the translocation of a
nucleotide through the
nanopore along with the anions carrying charges. Depending upon the polarity
of the voltage
difference, the nucleotide may be transported from the cis well to middle
well, or from the
middle well to the cis well. As the nucleotide transits through the nanopore,
the current across
the membrane may change due to nucleobase-dependent blockage of the nanopore
constriction.
[0180] In alternative examples, the polynucleotide does
not pass through the
nanopore, but tagged nucleotides are incorporated by a polymerase acting on
the
polynucleotide. In certain embodiments, a single-stranded polynucleotide, a
double-stranded
polynucleotide, tags or labels of incorporated nucleotides, or other
representatives of the
incorporated nucleotides, and any combination thereof may pass through the
nanopore. In
certain embodiments, tags or labels of incorporated nucleotides may be
separated or
dissociated from the polynucleotide, and such tags or labels may pass through
the nanopore
with or without the polynucleotide passing through the nanopore. Examples are
not limited to
how the polynucleotide communicates with the nanopore to cause signal
generation in the
nanopore sequencing device.
[0181) An electrical resistance Rp of the nanopore
(indicated as a resistor 223 in
FIG. 2) varies in response to the identity of one or more nucleobases near or
at the nanopore,
for example, while a nucleotide of the polynucleotide passes through the
nanopore, or while a
tagged nucleotide is being incorporated by a polymerase acting on the
polynucleotide, thus the
different tags of the tagged nucleotides change the resistance of the
nanopore. In some
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examples, as the polynucleotide enters the constriction of the nanopore, the
resistance Rp is
modulated based on the identity of the bases in the polynucleotide. In other
examples, the
resistance Rp is modulated based on the identity of a tag in the nanopore
constriction, while the
corresponding tagged nucleotide is being incorporated by a polymerase acting
on the
polynucleotide. In some examples, the resistance of the nanopore, Rp, changes
as a function
of the nucleobase at or near the nanopore and may range from about 0.5 to
about 5 giga-ohm
(Gi)). The resistance Rp may be relatively large and may vary by 30-40% as a
function of
different polynucleotide bases at or near the nanopore. In some examples, the
resistance Rp
may vary by between about 0.001% to about 1%, about 1% to about 5%, about 5%
to about
20%, about 20% to about 40%, about 40% to about 60%, or 60% to about 100%.
[0182]
In some examples, the nanochannel has a fixed, or substantially fixed
electrical resistance Re (indicated as a resistor 225 in FIG. 2). The
resistance Re of the
nanochannel is not modulated by the nucleoba se of the polynucleotide at or
near the nanopore.
In some examples, the resistance of the nanochannel, Re, may be about 1 to 5
giga-ohm (GO).
[0183]
The equivalent circuit 210 shown in FIG. 2 is a voltage divider, where
the
electrical potential of point 215 is the potential of the electrolyte in the
middle well. In certain
embodiments, the equivalent circuit of the nanopore sequencing device
satisfies the following
equations:
The potential Vm at point 215 given by
Vm = DV
(1)
where D is the voltage divider ratio
D = ¨
(2)
R, +
and V is the cis-trans bias.
[0184]
The electrical potential Vi of the electrolyte in the middle well
(indicated
as the voltage divider point 215 in FIG. 2) varies in response to the
variation in electrical
resistance Rp of the nanopore. Therefore, measuring the electrical potential
at the voltage
divider point 215 as the resistance Rp changes permits determination of the
resistance Rp, and
such information can be used to identify the nucleobases in the
polynucleotide. In some
examples, measuring the electrical potential at the voltage divider point 215
may be achieved
by coupling the sensing electrode to a voltage detector. In some examples,
measuring the
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electrical potential at the voltage divider point 215 may be achieved by
coupling an PET sensor
to the middle well. In one embodiment, the FET gate is coupled to the sensing
electrode, such
that the electrical potential of the voltage divider point 215 acts as the FET
gate potential and
establishes the FET operating point. Examples of measuring the response of the
FET include
measuring a source-drain current or measuring a potential at the source and/or
drain.
Additionally, a resistance of the FET channel can be measured to identify the
nucleobase in
the polynucleotide.
[0185] A method of using a nanopore sequencing device may
include introducing
an electrolyte into each of the cis well, the trans well, and the middle well.
After introducing
the electrolyte, the method may include providing a polynucleotide to be
sequenced into the
cis well. After providing the polynucleotide, the method may include applying
a voltage bias
between the cis electrode and the trans electrode. In some embodiments, the
voltage bias may
drive the polynucleotide from the cis well to the middle well, through the
nanopore As the
polynucleotide passes through the nanopore, the electrical resistance of the
nanopore varies in
response to an identity of nucleobases in the polynucleotide at the nanopore.
In alternative
embodiments, the polynucleotide does not pass through the nanopore, but tags
or labels of
nucleotides being incorporated by a polymerase acting on the polynucleotide
may pass through
the nanopore or may temporarily reside in the nanopore. Thus, the electrical
resistance of the
nanopore varies in response to an identity of the nucleotide being
incorporated, which is
complementary to the identity of a base in the polynucleotide. As a result,
the potential (Vi)
of the electrolyte in the middle well varies with the identities of bases in
the polynucleotide.
The potential (Vm) may be measured from the sensing electrode. The potential
(140 may be the
gate voltage applied to a FET, which modulates the conductivity of the FET
channel.
Therefore, measurements of the response of the FET can determine the identity
of the bases in
the polynucleotide.
[01861 In some embodiments of a nanopore sequencing
device, one or more trans
wells are fluidically connected to one or more cis wells by a plurality of
middle wells and the
respective nanopores and nanochannels. In various embodiments, the one or more
trans wells
may or may not be interconnected. In various embodiments, the one or more cis
wells may or
may not be interconnected. Each of the one or more trans wells may be
associated with a
respective trans electrode. In various embodiments, the trans electrodes may
or may not be
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operably connected to each other. Each of the one or more cis wells may be
associated with a
respective cis electrode. In various embodiments, the cis electrodes may or
may not be
operably connected to each other. In some embodiments, at least a group of
trans wells are
interconnected, at least a group of cis wells are interconnected, at least a
group of trans
electrodes are operably interconnected, at least a group of cis electrodes are
operably
interconnected, or any combination thereof.
[0187] In embodiments where a plurality of sequencing unit
cells forms an array
on a chip, each of the plurality of the sequencing unit cells in the array may
share a common
cis electrode and a common trans electrode. In another example, each of the
plurality of the
sequencing unit cells shares a common cis electrode, but has a distinct trans
electrode. In yet
another example, each of the plurality of the sequencing unit cells has a
distinct cis electrode
and a distinct trans electrode. In still another example, each of the
plurality of sequencing unit
cells has a distinct cis electrode and shares a common trans electrode In some
embodiments,
at least a group of sequencing unit cells in the array may share a common cis
electrode and a
common trans electrode. In some embodiments, at least a group of sequencing
unit cells shares
a common cis electrode, but each member of the group has a distinct trans
electrode. In some
embodiments, at least a group of sequencing unit cells shares a common trans
electrode, but
each member of the group has a distinct cis electrode.
[0188] FIG. 3A is a cross-sectional top view of the
nanopore sequencing device of
FIG. I A, showing an array 300A of sequencing unit cells. An array of straight
nanochannels
connect an array of middle wells to a trans well. For example, the cis well
314A connects to a
sequencing unit cell which includes a membrane 324A with a nanopore disposed
in it (not
shown), a middle well 315A, and a straight nanochannel 325A. The sensing
electrode 322A at
the bottom of the middle well 315A may be used to detect the electrical
potential of the
electrolyte in the middle well of this sequencing unit cell. The sequencing
unit cell then
connects to the trans well 316A. The effective width of the straight
nanochannel 325A is
narrowed by a deposited layer 337A. Also shown in FIG. 3A is the wall
structure 359A which
separates the individual sequencing unit cells.
[01891 FIG. 3B is a cross-sectional top view of the
nanopore sequencing device of
FIG. 1A having an alternative nanochannel structure. FIG. 3B shows an array
300B of
sequencing unit cells where an array of meandering/serpentine/tortuous
nanochannels connect
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an array of middle wells to a trans well. For example, the cis well 314B
connects to a
sequencing unit cell which includes a membrane 324B with a nanopore disposed
in it (not
shown), a middle well 315B, and a meandering nanochannel 325B. The sensing
electrode 322B
at the bottom of the middle well 315B may be used to detect the electrical
potential of the
electrolyte in the middle well of this sequencing unit cell. The sequencing
unit cell then
connects to the trans well 316B. The effective width of the meandering
nanochannel 325B is
narrowed by a deposited layer 337B. Also shown in FIG. 3B is the wall
structure 359B which
separates the individual sequencing unit cells. In the example shown in FIG.
3B, the
meandering nanochannel 325B has rectangular wave shape. Alternatively, the
meandering
nanochannel 325B may have a sine wave shape, a sawtooth shape, a zigzag shape,
a spiral
shape, or any combination thereof.
101901 FIG. 4 is a cross-sectional top view of an example
sequencing system 400
including the nanopore sequencing devices of FIG 1 A and inlet/outlet holes
which allow
fluidic and electric contact to cis/trans wells. For example, the cis well 414
connects to an array
of sequencing unit cells. A sequencing unit cell includes a membrane (an
example is the feature
labeled 424), a middle well (an example is the feature labeled 415), and a
nanochannel (an
example is the feature labeled 425). The membrane includes a nanopore disposed
in it (but not
shown). A sensing electrode (an example is the feature labeled 422) at the
bottom of a middle
well 415 may be used to detect the electrical potential of the electrolyte in
this middle well.
The sequencing unit cells connects to the trans well 416. The effective width
of a nanochannel
is narrowed by a deposited layer (an example is the feature labeled 437). Also
shown in FIG.
4 is the wall structure 459 which separates the individual sequencing unit
cells. To allow fluidic
or electrical contact and material exchange with external fluidic fixtures,
the cis well 414 are
connected with cis inlets/outlets 494 and the trans well 416 are connected
with cis trans/outlets
496.
101911 FIG. 8 is a cross-sectional top view of another
example sequencing system
800 including the nanopore sequencing devices of FIG. lA and inlet/outlet
holes which allow
fluidic and electric contact to cis well 866 and/or trans well 867. In some
embodiments, the
array comprises a single shared or common cis well 814. In some embodiments,
the array
comprises a shared or common trans well 816. A sequencing unit cell includes a
membrane
(an example is the feature labeled 824), a middle well (an example is the
feature labeled 815),
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and a nanochannel (an example is the feature labeled 825). In some
embodiments, the
nanochannel does not comprise a through-hole in the substrate. The membrane
includes a
nanopore disposed in it (but not shown). A sensing electrode (an example is
the feature labeled
822) at the bottom of a middle well 815 may be used to detect the electrical
potential of the
electrolyte in this middle well. The sequencing unit cells connects to the
trans well 816. To
allow fluidic or electrical contact and material exchange with external
fluidic fixtures, the cis
well 814 are connected with cis inlets/outlets 866 and the trans well 816 are
connected with
trans inlets/outlets 867.
[0192] In some embodiments, a nanopore sequencing device
may have a cis well
or cis channel position over the middle well. FIG. 9A shows a cross-sectional
side view of an
example nanopore sequencing device with the cis well on top of the device as
part of the fluidic
fixture and not integrated into the device. As shown in FIGS. 9A, 9B, and 9C,
in embodiments
900, the cis wells 914 are positioned so that membranes 924 are formed
horizontally parallel
to the horizontal nanochannel 925. In some embodiments, the nanochannel does
not comprise
a through-hole in the substrate. For example, in some embodiments, the
nanochannel 925 is
etched into a substrate. In some embodiments, the substrate comprises a
semiconductor wafer.
[0193] In some embodiment, the trans well may be formed in
the dielectric layer
of the first substrate, resulting in a trans well that is position vertically
with respect to the
middle well. FIG. 10 shows a cross-sectional side view of an example nanopore
sequencing
device with a common trans well 1016 fluidically connected to two middle wells
1015 through
respective nanochannels 1025. The trans well 1016 is formed in the substrate,
and is thus below
the substrate surface. The middle well 1015 is form in the patterned layer and
is thus above the
substrate. The nanochannel 1025 is form on the substrate surface, and it does
not contain a
through-hole in the substrate. In some embodiments, the cis well (not shown)
may be side-by-
side with and next to the middle well 1015, similar to the embodiment
illustrated in FIG. 1.A.
In other embodiments, the cis well may be positioned over the middle well
1016, similar to the
embodiment illustrated in FIG. 9A.
[0194] In a chip with an array of nanopore sequencing
devices, there may be one
common cis well and one common trans well communicating with a portion or all
of the
nanopore sequencing unit cells within the array in the chip. However, it
should be understood
that an array of the nanopore devices may also include several cis wells that
are fluidically
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isolated from one another and are fluidically connected to respective one or
more trans wells
fluidically isolated from one another. Multiple cis wells may be desirable,
for example, in
order to enable the measurement of multiple samples on a single chip. In some
embodiments,
a chip with an array of nanopore sequencing devices comprises one common cis
electrode, one
common trans electrode, one common cis well, one common trans well, and a
plurality of
nanopore sequencing devices, where each nanopore sequencing device can
separately measure
a single molecule of polynucleotide. In other embodiments, the chip with an
array of nanopore
sequencing devices comprises one common cis well, a plurality of trans wells,
and a plurality
of nanopore sequencing devices, where each nanopore sequencing device can be
individually
addressable with individual trans electrodes. In other embodiments, the chip
with an array of
nanopore sequencing devices comprises a plurality of cis wells, a plurality of
trans wells, and
a plurality of nanopore sequencing devices, where each nanopore sequencing
device can be
individually addressable with individual trans electrodes.
Additional Embodiments
[01951 FIG. 6 illustrates yet another example nanopore
sequencing device which
can generate bubbles via water electrolysis. Shown is an illustration of two
sequencing unit
cells laid out side-by-side and sharing the same trans well 616. A flowcell of
the nanopore
sequencing device is arranged between a first substrate and a second
substrate. The flowcell
may be filled with an electrolyte.
[0196] Above the second substrate, a top portion of the
nanopore sequencing
device may be connected to external fluidic fixtures. The cis well 614 is
connected to a
nanopore 623, in which a first nanoscale opening is formed. In some
embodiments, the
nanopore 623 may be formed in a protein 618 disposed into a membrane 624. In
some
embodiments, the membrane 624 may be arranged vertically with respect to the
first and
second substrates, on a side of the cis well 614. The nanopore 623 provides a
fluidic pathway
for an electrolyte to pass between the cis well 614 and the middle well 615.
The nanopore 623
fluidically communicates with a nanochannel 625 through the middle well 615.
The
nanochannel 625, in which a second nanoscale opening is formed, provides a
fluidic/ionic/electric pathway for the electrolyte/current to pass between the
middle well 615
and the trans well 616. The trans electrode may be operably connected to a
voltage supplier.
The flowcell includes the cis well 614, the trans well 616, a plurality of
middle wells 615 and
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their respective nanopores 623 and nanochannels 625, all of which are in
fluidic
communication.
[0197] The walls of the middle well 615 may be at least
partly defined by wall
structures 659. In some embodiments, the device may further include electrodes
to generate
gas bubbles 662 by electrolysis of the fluid in the nanocha.nnels to block the
liquid flow. For
example, in the event of rupture and/or failure of the membrane and/or
nanopore, gas bubbles
can be generated to block ionic current flow of the non-performing sequencing
unit cell so that
the other performing cis/trans cells may continue to properly perform.
[0198] A sensing electrode 622 may be arranged in the
first substrate and may be
exposed to the electrolyte in the middle well 615. The sensing electrode 622
may be used to
detect the electrical potential of the electrolyte in the middle well and to
transmit the detected
signal to a voltage detector circuit or a field effect transistor. In some
embodiments, the bottom
sensing electrode/FET 622 is used as one of the electrode pair, and the
horizontal nanochannel
625 which is metallized is used as the other electrode for electrolysis. Two
switches may be
added: (1) a first switch added to enable the bottom electrode/FET in a
sensing mode and (2)
a second switch added to enable the bottom electrode plus horizontal channel
electrode in
electrolysis mode.
[0199] FIG. 7A is a cross-sectional side view of an
example nanopore sequencing
device 700. The nanopore sequencing device 700 has the same structure as the
embodiment
shown in FIG. 6, except the bubble generating element is a micro heater or a
resistive heater
764. In FIG. 7A, the heating element 764 is underneath the nanochannel. The
heating element
may generate a bubble 762 within the nanochannel 725.
[0200] FIG. 7.B is a top view of another example nanopore
sequencing device 700.
The nanopore sequencing device 700 has the same structure as the embodiment
shown in FIG.
6, except two electrolysis electrodes 770 are located near the openings of the
nanochannel 725.
The electrodes can generate a bubble within the nanochannel.
Example Process of Manufacturing a Nan opore Sequencing Device
[0201] Some aspects of the present disclosure are directed
to methods of
manufacturing a nanopore sequencing device. In some embodiments, the method
comprises:
providing a first substrate comprising a dielectric layer and at least one
sensing electrode on a
surface of the first substrate; forming at least one nanochannel on the
surface of the first
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substrate; depositing a sacrificial filler layer in the at least one
nanochannel; depositing a
capping layer over the sacrificial filler layer; patterning the capping layer
to expose the at least
one sensing electrode and openings to the at least one nanochannel; and
removing the
sacrificial filler layer, thereby opening the at least one nanochannel. In
some embodiments, the
method of manufacturing the nanopore sequencing device comprises providing a
first substrate
comprising a dielectric layer and at least one sensing electrode on a surface
of the first
substrate; forming a trans well in the dielectric layer; and forming at least
one nanochannel on
the surface of the first substrate between the trans well and the at least one
sensing electrode.
In some embodiments, the nanochannel does not comprise a through-hole in the
substrate.
[02021 FIG. 5A to FIG. 5L illustrate an example
fabrication process flow of
manufacturing a nanopore sequencing device as disclosed herein.
[02031 In the process step shown in FIG. 5A, a first
substrate comprising a
dielectric layer and at least one sensing electrode is provided. The first
substrate includes a
CMOS wafer 557 formed of Si substrate manufactured with an integrated circuit,
for example
a sensing electrode 522 formed of Ru or TiN. The sensing electrode 522 may be
connected to
a voltage detector circuit, a FET sensor or an amplifier 5221. At least one of
the electrodes for
electrolysis, 5001 or 5002, may also be provided in this step. In alternative
embodiments,
flexible substrates such as ultrathin glass, metal foil, and plastic (polymer)
films may be used
for the bottom wafer, which may be manufactured with flexible electronics made
with organic
or carbon-based transistors.
[0204] In the process step shown in FIG. 5B, definition
and formation of the path
of the nanochannels 525 may be performed by lithography and etching
techniques.
[0205] In the optional process step shown in FIG. 5C,
reduction of the nanochannel
widths may be achieved by forming the deposited layer 537 via conformal
oxide/nitride layer
deposition.
[02061 In the process step shown in FIG. 5D, deposition of
a sacrificial filler layer
5001 may be performed to fill the nanochannels with sacrificial material. The
sacrificial layer
5001 may be formed of Ti or Al.
[02071 In the process step shown in FIG. 5E, planarization
of the sacrificial layer
may be performed by means of polishing. The sensing electrodes 522 is exposed,
while the
nanochannel path is filled with the sacrificial material. FIG. 5F shows a
cross-sectional top
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view of the nanochannels with the deposited sacrificial material and exposed
sensing
electrodes 522.
[0208] In the process step shown in FIG. 5G, passivation
of the wafer surface is
performed by deposition of a capping layer 547, which may be formed of
oxide/nitride. A
portion of the capping layer 547 may provide the base of the wall structure
147 in FIG. 1A. In
some embodiments, the passivation may also provide a hydrophilic cap for the
nanochannels.
[0209] In the process step shown in FIG. 5H, the capping
layer 547 is patterned to
expose the sensing electrode and the openings of the nanochannels to the
middle and trans
wells (yet to be formed). The nanochannels are now formed with the capping
layer closing the
top of the nanochannel path and having the two openings form on each end of
the nanochannel
path. This step may be performed by lithography and etching techniques.
102101 The process steps shown in FIGS. 5G and 5H may be
optional. If the
capping layer is not deposited after depositing the sacrificial material in
the nanochannel path,
the process step shown in FIG. 51 removal of the sacrificial material is
performed after the
process step shown in FIG. 5K.
[0211] In the process step shown in FIG. 51, removal of
the sacrificial material in
the nanochannels may be achieved by wet etch techniques. Once the sacrificial
material is
removed, the nanochannels are opened and will allow fluid communication
between the middle
well and the trans well to be formed in a later step.
[0212] In the process step shown in FIG. 5J, deposition of
a thick patterning
material layer 559 over the surface of the wafer is performed. A portion of
the patterning
material layer 559 may provide the wall structure 159 in FIG. IA. The
patterning material layer
559 may be formed of SIJ-8 photoresist or nanoimprint lithography (NIL)
resins, polyimide,
any kind of photo-patternable thick (spin-coated or laminated) resists such as
TMMF, TIVIMR,
silicones such as PDMS, thermoplastics such as PMMA, COC, PC (polycarbonate),
or any
suitable dielectric material.
[0213] In the process step shown in FIG. 5K, patterning
and etching of the
patterning material layer by photolithography or nanoimprint lithography
methods may form
a first patterned layer which in part defines the middle well and the trans
well, such as 515 and
516, respectively. In some embodiments, the first patterned layer may also in
part define the
cis wall, such as 514. In some embodiments, the trans well comprises a common
trans well.
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[0214] In the process step shown in FIG. 51,, a
complementary second substrate is
provided. The second substrate includes a top wafer 567 and a second patterned
layer of SII-8
or NM, resins. The top wafer 567 may further include fluidic inlet/outlet
holes, and a patterned
adhesive layer 549 formed of, e.g., photocurable resins, such as SU-8 or
benzocyclobutene
(BCB), or other suitable polymers, spin-on glasses, resists, and polyimides,
PUMS, fusion
bonding or covalent bonding of SiO2 surfaces, COC, methyl acrylic adhesive
(see
US20200009556A1, which is incorporated herein by reference). The patterned
adhesive layer
549 may provide the wall structure 149 in FIG. IA. The first patterned layer
of the bottom
wafer is aligned with the second patterned layer of the top wafer, and wafer
bonding of the
first patterned layer with the second patterned layer is performed via the
adhesive layer 549.
After wafer bonding, the cis, middle and trans wells are formed between the
bottom wafer and
the top wafer.
[0215] In the process step shown in FIG. 5M, membranes 524
may be introduced
between the cis wells and middle wells and may be arranged vertically. The
membrane has a
nanopore disposed in it to provide fluid communication between the cis well
and the middle
well. In some embodiments, proteins such as MspA. may be deposited into the
lipid membranes
to form nanopores through the membranes.
[0216] In some embodiments, the process step shown in FIG.
5L may not be
needed. For example, for embodiments where the cis well is over the middle
well shown in
FIG. 9A, a second substrate may not be required in some instances. In some
embodiments,
membranes 924 may be deposited horizontally over the middle well 915,
separating the middle
well 915 and the cis well 914.
[0217] In some embodiments, a nanopore sequencing device
as shown in FIG. 10
may be made by the following steps. Similar to the method described above, a
first substrate
including a dielectric layer and at least one sensing electrode 1022 on the
surface is provided.
The first substrate in these embodiments may have a thicker dielectric layer.
The trans well
1016 is formed by patterning and etching into the dielectric layer of the
first substrate. Thus
the trans well is below the surface of the first substrate.
[02181 Next a nanochannel 1025 is formed on the surface of
the first substrate
between the trans well 1016 and the sensing electrode 1025, which will provide
fluid
communication between the trans well 1016 and the middle well 1015 that will
be formed at a
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later step. A patterning material layer is then disposed over the first
substrate. For example, the
patterning material layer may be a dry film photoresist that is laminated over
the substrate. The
dry film photoresist may be any suitable photoresist material, including but
not limited to
TM.MF and SUS. The patterning material layer is subsequently patterned to form
a patterned
layer that includes a middle well 1015 positioned above the sensing electrode
1022 as
described above.
[0219] In some embodiments, the cis well may be formed in
the patterned layer,
resulting in embodiments where the cis well and the middle well being
positioned side-by-side.
In some embodiments, the cis well may be positioned over the middle well
similar to the
embodiment shown in FIG. 9A.
Additional Notes
102201 It should be appreciated that all combinations of
the foregoing concepts and
additional concepts discussed in greater detail below (provided such concepts
are not mutually
inconsistent) are contemplated as being part of the inventive subject matter
disclosed herein.
In particular, all combinations of claimed subject matter appearing at the end
of this disclosure
are contemplated as being part of the inventive subject matter disclosed
herein. It should also
be appreciated that terminology explicitly employed herein that also may
appear in any
disclosure incorporated by reference should be accorded a meaning most
consistent with the
particular concepts disclosed herein.
[0221] Reference throughout the specification to "one
example", "another
example", "an example", and so forth, means that a particular element (e.g.,
feature, structure,
and/or characteristic) described in connection with the example is included in
at least one
example described herein, and may or may not be present in other examples. In
addition, it is
to be understood that the described elements for any example may be combined
in any suitable
manner in the various examples unless the context clearly dictates otherwise.
[0222] It is to be understood that the ranges provided
herein include the stated range
and any value or sub-range within the stated range, as if such value or sub-
range were explicitly
recited. For example, a range from about 2 nm to about 20 nm should be
interpreted to include
not only the explicitly recited limits of from about 2 nm to about 20 nm, but
also to include
individual values, such as about 3.5 nm, about 8 nni, about 18.2 inn, etc.,
and sub-ranges, such
as from about 5 nm to about 10 nm, etc. Furthermore, when "about" and/or
"substantially"
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are/is utilized to describe a value, this is meant to encompass minor
variations (up to -F/- 10%)
from the stated value.
[0223] While several examples have been described in
detail, it is to be understood
that the disclosed examples may be modified. Therefore, the foregoing
description is to be
considered non-limiting.
I0224j While certain examples have been described, these
examples have been
presented by way of example only, and are not intended to limit the scope of
the disclosure.
Indeed, the novel methods and systems described herein may be embodied in a
variety of other
forms. Furthermore, various omissions, substitutions and changes in the
systems and methods
described herein may be made without departing from the spirit of the
disclosure. The
accompanying claims and their equivalents are intended to cover such forms or
modifications
as would fall within the scope and spirit of the disclosure.
[0225] Features, materials, characteristics, or groups
described in conjunction with
a particular aspect, or example are to be understood to be applicable to any
other aspect or
example described in this section or elsewhere in this specification unless
incompatible
therewith. All of the features disclosed in this specification (including any
accompanying
claims, abstract and drawings), and/or all of the steps of any method or
process so disclosed,
may be combined in any combination, except combinations where at least som.e
of such
features and/or steps are mutually exclusive. The protection is not restricted
to the details of
any foregoing examples. The protection extends to any novel one, or any novel
combination,
of the features disclosed in this specification (including any accompanying
claims, abstract and
drawings), or to any novel one, or any novel combination, of the steps of any
method or process
so disclosed.
[0226] Furthermore, certain features that are described in
this disclosure in the
context of separate implementations can also be implemented in combination in
a single
implementation. Conversely, various features that are described in the context
of a single
implementation can also be implemented in multiple implementations separately
or in any
suitable subcombination. Moreover, although features may be described above as
acting in
certain combinations, one or more features from a claimed combination can, in
some cases, be
excised from the combination, and the combination may be claimed as a
subcombination or
variation of a subcombination.
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[0227] Moreover, while operations may be depicted in the
drawings or described
in the specification in a particular order, such operations need not be
performed in the particular
order shown or in sequential order, or that all operations be performed, to
achieve desirable
results. Other operations that are not depicted or described can be
incorporated in the example
methods and processes. For example, one or more additional operations can be
performed
before, after, simultaneously, or between any of the described operations.
Further, the
operations may be rearranged or reordered in other implementations. Those
skilled in the art
will appreciate that in some examples, the actual steps taken in the processes
illustrated and/or
disclosed may differ from those shown in the figures. Depending on the
example, certain of
the steps described above may be removed or others may be added. Furthermore,
the features
and attributes of the specific examples disclosed above may be combined in
different ways to
form additional examples, all of which fall within the scope of the present
disclosure. Also,
the separation of various system components in the implementations described
above should
not be understood as requiring such separation in all implementations, and it
should be
understood that the described components and systems can generally be
integrated together in
a single product or packaged into multiple products. For example, any of the
components for
an energy storage system described herein can be provided separately, or
integrated together
(e.g.. packaged together, or attached together) to form an energy storage
system.
[0228] For purposes of this disclosure, certain aspects,
advantages, and novel
features are described herein. Not necessarily all such advantages may be
achieved in
accordance with any particular example. Thus, for example, those skilled in
the art will
recognize that the disclosure may be embodied or carried out in a manner that
achieves one
advantage or a group of advantages as taught herein without necessarily
achieving other
advantages as may be taught or suggested herein.
[0229] Conditional language, such as "can," "could,"
"might," or "may," unless
specifically stated otherwise, or otherwise understood within the context as
used, is generally
intended to convey that certain examples include, while other examples do not
include, certain
features, elements, and/or steps. Thus, such conditional language is not
generally intended to
imply that features, elements, and/or steps are in any way required for one or
more examples
or that one or more examples necessarily include logic for deciding, with or
without user input
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or prompting, whether these features, elements, and/or steps are included or
are to be
performed in any particular example.
[0230] Conjunctive language such as the phrase "at least
one of X, Y, and Z,"
unless specifically stated otherwise, is otherwise understood with the context
as used in general
to convey that an item, term, etc. may be either X, Y, or Z. Thus, such
conjunctive language
is not generally intended to imply that certain examples require the presence
of at least one of
X, at least one of Y, and at least one of Z.
[0231] Language of degree used herein, such as the terms
"approximately,"
"about," "generally," and "substantially" represent a value, amount, or
characteristic close to
the stated value, amount, or characteristic that still performs a desired
function or achieves a
desired result.
[0232i The scope of the present disclosure is not intended
to be limited by the
specific disclosures of preferred examples in this section or elsewhere in
this specification, and
may be defined by claims as presented in this section or elsewhere in this
specification or as
presented in the future. The language of the claims is to be interpreted
broadly based on the
language employed in the claims and not limited to the examples described in
the present
specification or during the prosecution of the application, which examples are
to be construed
as non-exclusive.
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